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Conventional kinesin KIF5B mediates insulin‐stimulated GLUT4 movements on microtubules

Conventional kinesin KIF5B mediates insulin‐stimulated GLUT4 movements on microtubules The EMBO Journal Vol. 22 No.10 pp. 2387-2399, 2003 Conventional kinesin KIFSB mediates insulin­ stimulated GLUT4 movements on microtubules mechanism whereby insulin signaling causes intracellular Sabina Semiz, Jin G.Park, membranes containing GLUT4 to move and fuse with the Sarah M.C.Nicoloro, Paul Furcinitti, plasma membrane requires the p85/p 110 type phos­ Chuanyou Zhang, Anil Chawla, John Leszyk phatidy linositol 3 (PI3)-kinase (Cheatham et al., 1994; and Michael P.Czech Okada et al., 1994; Sharma et al., 1998) and appears to Program in Molecular Medicine, 373 Plantation Street, University of involve the downstream protein kinase Akt2 (Hill et al., Massachusetts Medical School, Worcester, MA 01605, USA 1999; Cho et al., 2001). Some components involved in Corresponding author membrane trafficking of GLUT4 have also been identified, e-mail: [email protected] including the SNARE proteins V AMP2 (Cheatham et al., 1996; Martin et al., 1996) and syntaxin-4 (Volchuk et al., S.Semiz and J.G.Park contributed equally to this work 1996; Olson et al., 1997; Pessin et al., 1999), as well as SNAP25 (Jagadish et al., 1996) and SNAP23 (Kawanishi Insulin stimulates glucose uptake in muscle and adi­ et al., 2000). In both primary and cultured adipocytes, pose cells by mobilizing intracellular membrane vesicles containing GLUT4 glucose transporter GLUT4 recycles slowly between intracellular membranes proteins to the plasma membrane. Here we show in and the plasma membrane even in the absence of insulin, live cultured adipocytes that intracellular membranes and recycling is markedly enhanced by activation of containing GLUT4-yellow fluorescent protein (YFP) GLUT4 exocytosis by the hormone (Jhun et al., 1992; move along tubulin--cyan fluorescent protein-labeled Yang and Holman, 1993). A PI3-kinase-dependent step in microtubules in response to insulin by a mechanism this process may be fusion of GLUT4-containing mem­ that is insensitive to the phosphatidylinositol 3 (PI3)­ branes with the plasma membrane, based on the identifi­ kinase inhibitor wortmannin. Insulin increased by cation of the insulin-regulated protein synip as an apparent several fold the observed frequencies, but not veloci­ modulator of syntaxin-4 function (Min et al., 1999). ties, of long-range movements of GLUT4-YFP on Evidence has been steadily accumulating in favor of the microtubules, both away from and towards the peri­ hypothesis that GLUT4 movements in adipocytes and nuclear region. Genomics screens show conventional muscle cells are also dependent on cytoskeletal structures. kinesin KIFSB is highly expressed in adipocytes and Many laboratories have confirmed that optimal GLUT4 this kinesin is partially co-localized with perinuclear translocation in response to insulin requires intact actin GLUT4. Dominant-negative mutants of conventional filaments (Wang et al., 1998; Omata et al., 2000; Bose kinesin light chain blocked outward GLUT4 vesicle et al., 2001; Emoto et al., 2001; Patki et al., 2001; Jiang movements and translocation of exofacial Myc-tagged et al., 2002), and co-localization of GLUT4 and GLUT4-green fluorescent protein to the plasma mem­ polymerized actin is observed in insulin-treated muscle brane in response to insulin. These data reveal that cells (Asahi et al., 1999; Khayat et al., 2000) and insulin signaling targets the engagement or initiates adipocytes (unpublished data). We found that depolymer­ the movement of GLUT4-containing membranes on ization of intermediate filaments and microtubules also microtubules via conventional kinesin through a PI3- disrupted perinuclear GLUT4 localization, as well impair­ kinase-independent mechanism. This insulin signaling ing GLUT4 responsiveness to insulin (Guilherme et al., pathway regulating KIFSB function appears to be 2000; Emoto et al., 2001). Numerous laboratories have required for GLUT4 translocation to the plasma confirmed that depolymerization of microtubules partially membrane. inhibits GLUT4 recycling and insulin-mediated GLUT4 Keywords: adipocytes/GLUT4/insulin/kinesin/ translocation (Fletcher et al., 2000; Emoto et al., 2001; microtubules Molero et al., 2001; Olson et al., 2001). Live cell imaging of 3T3-Ll adipocytes expressing GLUT4-green fluores­ cent protein (GFP) has revealed short sporadic GLUT4 movements, as well as longer range movements, that are Introduction disrupted by colchicine (Fletcher et al., 2000). However, Glucose homeostasis in humans is dependent upon the while there is agreement that endocytosis of GLUT4 to catalytic activity of a glucose transporter protein, GLUT4, perinuclear membranes is driven by dynein motors on that is highly expressed in fat and muscle and responds to microtubule tracks, a recent report has questioned the insulin by translocation from intracellular vesicles to the involvement of microtubules and kinesins in the outward plasma membrane (Olson and Pessin, 1996; Czech and process (Molero et al., 2001). Another report concludes Corvera, 1999; Bryant et al., 2002). Ablation of GLUT4 outright that microtubules are not required for GLUT4 expression in either adipocytes (Abel et al., 2001) or translocation (Shigematsu et al., 2002). Thus, the issue of skeletal muscle (Zisman et al., 2000) of mice can lead to microtubule and kinesin involvement in insulin-mediated glucose intolerance, insulin resistance and diabetes. The GLUT4 movements has remained unresolved. © European Molecular Biology Organization 2387 S.Semiz et al. The aim of this study was to visualize directly GLUT4- suggest that an individual GLUT4-YFP-containing yellow fluorescent protein (YFP) movements in live vesicle may be bound to and carried by both minus end­ insulin-sensitive 3T3-Ll adipocytes under conditions and plus end-directed motor proteins. where microtubules could also be observed. Here we report images showing fluorescence from GLUT4-YFP Insulin signaling increases the frequency of moving along microtubules labeled with tubulin--cyan long-range GLUT4-YFP movements fluorescent protein (CFP), providing direct evidence that Experiments were conducted to determine whether insulin membranes containing GLUT4 move on microtubule action modulates long-range movements of GLUT4-YFP­ tracks. Remarkably, insulin treatment is shown to increase containing vesicles on microtubules. Analysis of numer­ the frequency of these long-range movements both away ous movies of control versus insulin-treated 3T3-Ll from and towards the perinuclear region. The outward adipocytes expressing both GLUT4-YFP and tubulin­ movements of GLUT4 towards the plus ends of micro­ CFP revealed no significant effect of the hormone on the tubules appear to be physiologically relevant because mean velocities of GLUT4-YFP movements in either these movements as well as insulin-mediated GLUT4 inward or outward directions (Figure 3A). There was also translocation to the plasma membrane was blocked by no consistent change in the distribution of velocities dominant inhibitory kinesin light chain proteins. These exhibited by various groups of GLUT4-YFP-containing new data are consistent with the hypothesis that GLUT4- vesicles (Figure 3B). However, insulin signaling in these containing membranes are cargo for conventional kinesin, cultured adipocytes caused a marked increase in the which moves these membranes towards the cell periphery observed frequency of total long-range movements of in response to insulin. GLUT4-YFP on microtubules (Figure 3C). This increase in the number of such events per unit time was highly significant when either inward ( ~ 3-fold increase) or Results outward ( ~8-fold increase) directions were monitored. GLUT4-containing membranes move on Thus insulin-treated adipocytes display much higher numbers of GLUT4-YFP-containing vesicles engaged in microtubules Preliminary imaging performed on live 3T3-Ll adipocytes extended linear motion along microtubules than untreated expressing GLUT4-GFP or GLUT4-YFP confirmed two cells at any given time. earlier reports that showed very active movements of To further confirm that these long-range movements fluorescent vesicles which were mostly randomly directed represent microtubule-based GLUT4-YFP transport, and short range (Oatey et al., 1997; Patki et al., 2001). microtubules were depolymerized by treatment with Occasional linear movements over distances of 10 µm or colchicine. As expected, the colchicine treatment resulted more could be observed as well, as noted previously in the loss of our ability to visualize microtubules with (Oatey et al., 1997). To examine whether these long-range tubulin-CFP (unpublished data) and the abolition of movements are microtubule based, 3T3-Ll adipocytes insulin-stimulated long-range GLUT4-YFP movements electroporated with plasmids encoding both GLUT4-YFP (Figure 3C). Interestingly, very active short-range random and tubulin--CFP were examined by time-lapse micro­ movements of GLUT4-YFP-containing vesicles could scopy at a single optical plane. Under these conditions, still be observed in the periphery of the colchicine-treated complex networks of labeled microtubules could be cells, which may represent movements on other cyto­ visualized throughout the cultured adipocytes (Figure 1). skeletal structures, such as F-actin. These results demon­ Remarkably, the majority of the long-range GLUT4-YFP­ strate the specific requirement of intact microtubules for containing vesicle movements occurred along the micro­ long-range movements of GLUT4-YFP in cultured tubule tracks within this network (Figures 1 and 2). adipocytes in response to insulin. Figure 1 shows a typical single long-range movement of GLUT4-YFP fluorescence over a distance of ~10 µm in Insulin signaling to increase long-range 40 s. Moreover, some vesicles could be observed to travel movements of GLUT4-YFP on microtubules is over a total distance of 20 µm by transiting onto several Pl3-kinase independent different microtubules. These data directly demonstrate It is likely that there are multiple steps in the regulated that the long-range movements of GLUT4-YFP vesicles movements and fusion of GLUT4-containing membranes are associated with microtubule-based motor activity with the plasma membrane. These steps might include rather than being random, Brownian or based on other membrane budding or release of membranes from tether­ cytoskeletal components. ing in the perinuclear region, movement along micro­ Using the microtubule organizing center (MTOC) and/ tubules (Figures 1-3), movement along actin filaments or the nuclei as visual indicators, we were able to (Omata et al., 2000; Bose et al., 2001; Jiang et al., 2002), distinguish the directionality of many of the GLUT4- and docking and fusion with the plasma membrane YFP movements. Figure 2 shows three examples in which (Cheatham et al., 1996; Martin et al., 1996; Volchuk the identical GLUT4-YFP-containing vesicles were cap­ et al., 1996; Min et al., 1999). Insulin regulation of this able of moving both towards (right panels) and away from overall process resulting in increased GLUT4 proteins in (left panels) the perinuclear region of the adipocyte. These the plasma membrane and enhanced glucose transport into directions are referred to as 'inward' and 'outward', adipocytes is totally inhibited by agents that disrupt PI3- respectively, in Figure 2. Analysis of the velocities of kinase (Cheatham et al., 1994; Okada et al., 1994; Sharma these long-range movements in many cells (Figures 2 and et al., 1998). This could mean that: (i) all the above 3A) showed that the speeds of outward and inward regulated steps depend on PI3-kinase; (ii) as few as one movements were not significantly different. These data step in the pathway exhibits such dependency; or (iii) none 2388 KIF5B mediates GLUT4 movements on microtubules Fig. 1. GLUT4-YFP vesicles move on microtubules in cultured adipocytes (a time-lapse movie is presented in the Supplementary data available at The EMBO Journal Online). (A) 3T3-Ll adipocytes were electroporated with plasmids encoding GLUT4-YFP and tubulin-CFP. After serum starva­ tion, cells were treated with 100 nM insulin for 15 min, followed by time-lapse image recording with a 5 s interval up to 15 min. Shown here is a merged image of the first and the last frames of a 40 s sequence, showing that a GLUT4-YFP-containing vesicle (red) adjacent to microtubule (green) moves from right to left as indicated by arrows with time points (seconds). N, nucleus. (B) Nine sequential frames of boxed area in (A) are enlarged and merged. Positions of GLUT4-YFP vesicles are indicated by arrows with elapsed time (seconds). of the regulated steps are PI3-kinase dependent, but part of (Figure 3A) or the distribution of the velocities among the machinery of forming the regulatable sequestration vesicle populations (Figure 3B). compartment or other aspect of the cell machinery Based on this failure of wortmannin to inhibit linear necessary for the response is PI3-kinase dependent. In GLUT4-YFP movements, we wanted to confirm the order to examine whether insulin regulates linear GLUT4- known effect of the drug to inhibit GLUT4 translocation YFP movements on rnicrotubules through a PI3-kinase­ to the plasma membrane in response to insulin under these dependent mechanism, we treated 3T3-Ll adipocytes with same experimental conditions (Figure 4). Using a wortmannin prior to addition of insulin. Under the GLUT4-GFP construct with a Myc tag on the first conditions of these experiments wortmannin abolished exofacial loop as reporter, it was found that the insulin­ the strong increase in phosphorylation of the protein stimulated increase in cell-surface Myc-GLUT4-GFP was kinase Akt in response to insulin (data not shown). indeed nearly, but not completely, inhibited (74%) by Surprisingly, this treatment did not affect the number of 100 nM wortmannin, similar to a previous report long-range GLUT4-YFP movements per unit time in (Hausdorff et al., 1999). Interestingly, however, 46% of insulin-treated cells (Figure 3C), the average maximal these wortmannin- and insulin-treated cells did display a velocities in either inward or outward GLUT4 movements strong rim of GLUT4-GFP fluorescence around the cell 2389 S.Semiz et al. Inward Outward 0.328 µm sec· 1 0.346 µm sec· 1 0.160 µm sec·1 0.392 µm sec· 1 0.164 µm sec· 0.272 µm sec· 1 Fig. 2. A single GLUT4-YFP vesicle moves bi-directionally on microtubules. Three different GLUT4-YFP vesicles from time-lapse images that displayed bi-directional movements are shown. The movements were categorized as 'inward' and 'outward', as described in the text. The numbers inside images indicate elapsed time (seconds) from the first frame of each sequence. Right and left panels show bi-directional movements of the identical vesicles in the same field of cells. The numbers below images indicate maximal velocity of vesicles. Bars, 4 µm. periphery (Figure 4B). These data are consistent with the hypothesis suggests that the fusion step whereby Myc­ hypothesis that insulin acts to move GLUT4-containing GLUT4-GFP is inserted into the plasma membrane to vesicles towards the cell surface on rnicrotubules in the become accessible to Myc antibody in unpermeabilized cells requires PB-kinase. presence of wortmannin. According to this model, GLUT4-containing vesicles in cells treated with insulin plus wortmannin can then dock with the plasma mem­ Insulin-stimulated GLUT4 translocation requires brane, leading to the rim of Myc-GLUT4-GFP at the sub­ conventional kinesin KIF58 plasma membrane region observed by monitoring the GFP Plus end-directed transport of vesicles and organelles on signal but not the exofacial Myc signal (Figure 4A). Some rnicrotubules is mediated by the large family of kinesin of these vesicles are perhaps returned to the perinuclear motor proteins. In a screen for genes highly expressed in region, while others accumulate as docked vesicles. This 3T3-Ll adipocytes using Affymetrix GeneChip arrays 2390 KIF5B mediates GLUT4 movements on microtubules GFP aMyc A A 0.6 ..__ Insulin □ Basal - - � - ■ Insulin ■ Wort + Insul in - - - - f-- - -- -- Inward Outward Wort+ Insulin �4 □ Basal +- -- �1-- --i ■ Insulin -- ------- --- �3 ■ Wort + Insulin � 2 G1 !o 0,2 0.4 0.6 M 0.2 0. 0.6 0.8 .. Inward velocity Outward velocity 1 1 (µm sec· ) f ,m aec· ) c:: 1.4 • *" □ Basal nr::7 1.2 - - -+ -- -- - --1 ■ Insulin Wort+ Insulin ----- 1.0 1-- --1 l2l Col + Insulin 1ia 0B 0.6 GI 0 , 4 0.2 "" 0.0 Fig. 4. Accumulation of GLUT- YFP-containing vesicles near the cell Total Inward Outward periphery in adipocytes treated with wortrnannin plus insulin. (A) 3T3- Ll cells were electroporated with the Myc-GLUT4-EGFP plasmid, starved, and then either treated with wortrnannin for 15 min and then Fig. 3. Insulin increases the number, but not velocities, of long-range 100 nM insulin for 30 min (Wort + Insulin), or 100 nM insulin alone GLUT4-YFP vesicle movements on microtubules in a wortmannin­ for 30 min (Insulin). Cells were then fixed and stained for plasma mem­ insensitive manner. 3T3-Ll adipocytes expressing GLUT4-YFP and brane GLUT4 (red) with anti-Myc antibody and rhodamine-conjugated tubulin-CFP were starved and either left untreated (Basal), or treated secondary antibody without permeabilization. EGFP signal is shown in with either 100 nM insulin (Insulin), 100 nM wortrnannin for 15 min green. Bar, 10 µm. (B) Myc-GLUT4-EGFP-electroporated 3T3-Ll followed by a 15 min insulin treatment (Wort + Insulin), or 50 µM adipocytes were starved and left untreated (Basal), treated with either colchicine for 2 h followed by a 15 min insulin treatment (Col + 100 nM wortmannin for 45 min (Wort) or 100 nM insulin for 30 min Insulin). Long-range movements of GLUT4-YFP vesicles from 6 to 16 (Insulin), or pre-treated with 100 nM wortrnannin for 15 min followed time-lapse images for each condition were analyzed for their velocities by a 30 min 100 nM insulin treatment (Wort + Ins). For each set, 50 and number of events. (A) Average maximal velocities of GLUT4- cells were counted for the EGFP and the Myc rims. YFP vesicles moving either towards (inward) or away from (outward) the perinuclear region. Total vesicle counts represent the sum of all long-range GLUT4-YFP vesicle movements observed, including those where directionality could not be unequivocally determined. Error bars adipocytes compared with fibroblasts was observed by represent the SEM. (B) Distribution of maximal velocities of GLUT4-YFP vesicles. (C) The number of GLUT4-YFP vesicle move­ western blot analysis (Figure 5A). Conventional kinesin ments in a unit area is shown. Error bars represent the SEM. *P = KIF5B is composed of two heavy chains and two light 0.0006; **P = 0.002; #P = 0.01; ##P = 0.004. chains in a heterotetrameric configuration (Figure 6A) and is expressed in many tissues, while the other conventional kinesin isotypes KIF5A and KIF5C function mainly in (U74 series), oligonucleotides derived from the gene neuronal tissues (Goldstein and Yang, 2000). Since the sequence of the conventional kinesin heavy chain KIF5B anti-KIF5 antibody (H2) we used recognizes all three were found to yield the highest signal of all the kinesins isotypes but has lowest affinity for KIF5B (Kanai et al., represented on the arrays (Table I). Furthermore, higher 2000; Cai et al., 2001), we performed immunoprecipit­ expression of conventional heavy chain in cultured ation with the H2 antibody followed by mass spectrometry 239 1 439 S.Semiz et al. meric kinesin complex and block the kinesin--cargo Table I. Expression of kinesins in 3T3-Ll adipocytes was analyzed binding. Both the HA-KLC-L176 and HA-KLC-TPR6 by Affymetrix GeneChips constructs have been shown to disrupt conventional Kinesin type Average SEM Chip Probe set kinesin function when expressed in other cell types signal set (Verhey et al., 2001). We also employed expression of KIF5B 20 732 2220 B 113588_f_at the native kinesin light chain in these experiments as a KIF21B 4063 666 B 11482l_at control. After electroporation with these HA- or CFP­ KIF3B 3520 996 B 115208_at conjugated constructs, 3T3-Ll adipocytes remained intact KIF3A 2829 334 B 115929_at and showed no phenotypic defect. All three KLC KIF21A 204 1 572 B 11723l_at KIFlB 1579 192 B 115895_at constructs localized mostly in the perinuclear region, KIF1 3A 853 334 B 109305_at while two dominant-negative KLC mutants often mis­ KIF2 574 168 A 99962_at localized into the nucleus upon higher expression (Figures KIF3C 377 303 A 93635_at 6B, and 7 A and B). Consistent with the known function of KIF4 274 42 A 104644_at KIFC2 123 68 A 9389l_at conventional kinesin in mitochondrial dispersion (Tanaka KIFC3 73 105 A 104335_at et al., 1998), cells expressing the dominant-negative KLC KIFlA 52 119 A 92890_at mutants displayed reduced mitochondrial staining in the KIFCl -978 461 A 98471_f_at peripheral cytoplasm (Figure 6B). Confirming the speci­ KLCl 786 90 A 93565_at ficity of the dominant-negative mutant function, expres­ KLC2 242 A 102636_at sion of either the full-length or the dominant-negative KLC proteins did not affect intermediate filament distri­ Shown here are the average signal representing the mean of average bution (data not shown) or insulin-stimulated cortical actin differences and the SEM from three independent experiments. The names of murine genome chip sets (U74A and U74B) and individual rearrangement (Figure 6B). To test whether vesicle probe sets giving the highest signal for a particular kinesin are shown. movements stimulated by insulin are mediated by Expression signals of KLCl and KLC2 are also presented. KIF5B, 3T3-Ll adipocytes were electroporated with the plasmid containing CFP-conjugated KLC constructs and GLUT4-YFP, and counted for the number of vesicle in order to confirm the expression of KIF5B. As shown in movements toward the cell periphery by live cell imaging. Figure 5B and C, a single major band was detected at Figure 6C showed that the dominant-negative KLC ~120-130 kDa after an NP-40 extraction in both starved mutants, but not the full-length KLC, significantly and insulin-stimulated conditions. Mass spectrometry inhibited insulin-stimulated outward long-range move­ analysis of the band identified peptides with sequences ments of GLUT4-containing vesicles. Together with high that exactly match the murine kinesin heavy chain KIF5B, expression of KIF5B (Figure 5), these data suggest that indicating that only this isotype is present in 3T3-Ll KIF5B is required for mobilizing GLUT4-containing adipocytes (Figure 5C). Experiments were therefore vesicles in response to insulin in 3T3-Ll adipocytes. designed to test the hypothesis that conventional kinesin We next tested whether these outward GLUT4-contain­ KIF5B function is required for GLUT4 translocation to ing vesicle movements mediated by KIF5B are required the plasma membrane in response to insulin. First, the for insulin-stimulated translocation of GLUT4 to the intracellular localizations of endogenous conventional plasma membrane. As shown in Figure 7 A, expression of kinesin and GLUT4 were analyzed in 3T3-Ll adipocytes either dominant-negative mutant KLC abolished the by immunostaining. The kinesin heavy chain is mainly ability of insulin to stimulate translocation of exofacial present in the perinuclear region of 3T3-Ll adipocytes and Myc-GLUT4-GFP to the cell surface, as reflected by the partially co-localizes with GLUT4-containing vesicles absence of anti-Myc antibody binding to adipocytes under (Figure 5D). These data are consistent with the hypothesis these conditions. In contrast, intact adipocytes expressing that this kinesin is associated with intracellular GLUT4- Myc-GLUT4-GFP and the native kinesin light chain containing membranes in intact cultured adipocytes. displayed strong anti-Myc antibody binding to the cell Next we employed two dominant inhibitory kinesin surface in response to insulin (Figure 7 A and B). light chain mutant proteins, based on the KLCl isoform, to Quantitation of the results from a large number of disrupt conventional kinesin KIF5B function (Figure 6A). adipocytes in these experiments was performed by visually The KLCl isoform was found to be expressed in counting the number of cells that display cell-surface adipocytes, while KLC-2 is not (Table I). One of the binding of anti-Myc antibody (Figure 8A), as well as by constructs, hemagglutinin (HA)-tagged KLC-TPR6 (or quantifying the ratio of signal intensity from the anti-Myc CFP-KLC-TPR6), lacks the heptad repeats domain that antibody binding (cell-surface Myc-GLUT4-GFP) versus functions as the kinesin heavy chain binding region the signal from the total GFP fluorescence (total expressed (Diefenbach et al., 1998; Verhey et al., 1998; Kamal and Myc-GLUT4-GFP) (Figure 8B). These techniques for Goldstein, 2002). Its expression is predicted to block estimating the action of insulin on translocation of Myc­ interaction between endogenous kinesin and cargo GLUT4-GFP have been described in detail previously proteins by saturating kinesin receptors such as those (Park et al., 2001; Jiang et al., 2002). By both these that might be present in GLUT4-containing vesicles. The measurements, expression of the inhibitory kinesin mutant other construct, HA-tagged KLC-Ll 76 (or CFP-KLC­ proteins exerted virtually complete abolition of cell-surface Ll 76), lacks the C-terminal TPR6 domain involved in Myc-GLUT4-GFP display in response to insulin (Figures 7 KLC--cargo binding (Skoufias et al., 1994; Bi et al., 1997). and 8), consistent with a requirement of conventional This mutant is predicted to incorporate into heterotetra- kinesin for insulin action on GLUT4 translocation. 2392 KIF5B media tes GLUT4 movements on microtubule s IP : lgG H2 Ad i Fib B kDa Basal Insulin Basal lnsur in 175- 17 5 - KHC 83- 62- .-KHC 18: H2 47- 2 · lg 25- IP: H2 Ne Insulin Lysa1e Sl iver stain aKHC aG LUT4 Merg e Fig. 5. Endogenous conventional kinesin KIF5B partially co-localizes with GLUT4 in 3T3-Ll adipocytes. (A) Confluent fibroblasts (Fib) and fully diff erentiated 3T3-Ll adipocytes (Adi) were analyzed by irnrnunoblotting using anti-kinesin heavy chain H2 antibodies. (B) Starved (Basal) and insulin-stimulated 3T3-Ll adipocytes were lysed and subject to irnrnunoprecipitation (IP) with either non-immune mouse IgG (lgG) and anti-kinesin heavy chain H2 antibody. lg, immunoglobulin. (C) A gel was prepared as above and silver stained. The 120 kDa band was excised and analyzed by mass spectrometry. Shown are two peptides for which the amino acid sequences were matched to the murine conventional kinesin heavy chain KIF5B. (D) 3T3-Ll adipocytes were immunostained with anti-kinesin heavy chain H2 antibody (aKHC; red) and anti-GLUT4 antibody (aGLUT4; green). Two different cells are shown with merged images (Merge) of both channels. Bars, 10 µm. 2393 S.Semiz et al. A C c 1. 0 �---------------� (·) 1 o.e ] Globu lar ta il 8 � 0.6 +----------- Corled-coil I 0.4 - -- ii 0.2 -t- -,.....-=1=----.=!=;- -1 ... TPR motifs 0.0 KLC Ba.sal Insul in HA 176 L1 76 TPR6 Insulin KLC Basal L1 76 TPR6 aHA Actin aHA Mita Fig. 6. Dominant-negative KLC mutants inhibit insulin-stimulated GLUT4-YFP vesicle movements. (A) Top diagram shows heterotetrameric structure of conventional kinesin and a proposed model for cargo binding (MT, microtubule). Structures of wild type (KLC), and C-terminal (Ll76) and an N-terminal deletion (TPR6) mutants of kinesin light chain are shown in the bottom diagram. All constructs have HA tags within their N-termini. The numbers represent amino acid sequences. These diagrams are adapted from Verhey et al. (2001). (B) Plasmids containing the full-length (KLC) and deletion mutant (Ll 76 and TPR6) constructs were electroporated into 3T3-Ll adipocytes. Cells were starved and either left untreated (Basal) or stimulated with insulin for 30 min. Electroporated cells were identified by irnmunostaining with anti-HA antibody ( cdIA). F-actin and mitochondria (Mito) were visualized with rhodarnine-phalloidin and Mitotracker, respectively. Bar, 10 µm. (C) 3T3-Ll adipocytes were electroporated with plasmids containing GLUT4-YFP and the kinesin light chain-CFP constructs. The number of GLUT4-YFP vesicles moving away from the nucleus in either basal or insulin-stimulated cells was counted. Five to seven cells were counted for each condition. Error bars represent the SEM. *P = 0.019; **P = 0.016. ■ KLC ■ L176 0 TPR8, ....._________.� KIF5B mediates GLUT4 movem ents on microtubules aHA GFP-G LUT4 nMyc aHA aMyc KlC ·. ·• (-) L176 Basal KLC TPR6 KLC L1 76 L176 In sulin . TP R6 TPR6 Fig. 7. Dominant-negative kinesin light chain mutants block insulin-stimulated GLUT4 translocation to the cell surface. (A) 3T3-Ll cells were electro­ porated with the Myc-GLUT4-EGFP plasmid and plasmid containing either the wild-type or dominant-negative KLC constructs. Cells were starved and either left untreated (Basal) or incubated with 100 nM insulin (Insulin), and immunostained for the plasma membrane-fused GLUT4 with anti­ Myc antibody (cxMyc) without permeabilization. Cells were then permeabilized and immunostained with anti-HA antibody (o;HA). Low magnification images show single-transfected (Myc-GLUT4-EGFP) cells and double-transfected (GLUT4-Myc-EGFP and KLC constructs) cells in the same viewing fields. Bars, 20 µm. (B) High magnification images of insulin-treated 3T3-Ll adipocytes that were either single-(-) or double-transfected as described in (A). Bars, 10 µm. Discussion translocation (Guilherme et al., 2000; Emoto et al., 2001). The present findings directly establish the conclusion that GLUT4-containing membranes move on microtubules in Long-range transport of GLUT4 vesicles on an outward direction towards the periphery of cultured microtubules adipocytes, as well as in an inward direction towards the A key finding of this study is the observed movement of perinuclear region. fluorescence signal from GLUT4-YFP along tracks delineated by microtubules labeled with tubulin-CFP (Figures 1 and 2). Furthermore, our data document such KIF58 mediates GLUT4 vesicle movements on linear movements of GLUT4-YFP over distances as long microtubules as 20 µm, directed towards both the plus and minus ends of A second significant finding reported here is the identifi­ cation of conventional kinesin KIFSB as a molecular microtubules (Figures 2 and 3). Remarkably, insulin motor required for GLUT4 translocation in response to stimulates the number of these events observed per unit time in cultured adipocytes (Figure 3). Depolymerization insulin (Figures 7 and 8). Conventional kinesin was first of microtubules by colchicine results in complete inhib­ identified as a processive molecular motor involved in ition of long-range GLUT4-YFP-containing vesicle driving vesicles and organelles in axons of vertebrate and movements (Figure 3C). Only a relatively small number squid brain (Brady, 1985; Vale et al., 1985). Kinesin heavy of GLUT4 vesicles can be seen traveling over long chain has a microtubule plus end-directed motor domain, distances after insulin stimulation relative to the total while kinesin light chain is generally thought to be number of GLUT4-containing membranes observed in a involved in kinesin binding to cargos, mediating their single field. However, our live cell microscopy is motility toward the cell periphery (Hirokawa, 1993; performed at a single optical plane, which probably Skoufias and Scholey, 1993). Recent reports demonstrated provides an underestimate of the total events per cell. that conventional kinesin is essential for mitochondrial These data extend previous indirect evidence suggesting (Tanaka et al., 1998), lysosomal (Tanaka et al., 1998) and that GLUT4-containing membranes are mobile on micro­ endosomal (Nielsen et al., 1999) transport, as well as for tubules based on the distance and linearity of their the microtubule-based motility of virus particles in movements (Oatey et al., 1997) as well as on the inhibitory infected cells (Rietdorf et al., 2001). Of the three isotypes actions of microtubule depolymerizing agents on GLUT4 of conventional kinesin known, only KIF5B could be 239 5 S.Semiz et al . detected in adipocytes. Using the same dominant inhibi­ 1. 0 tory kinesin light chain constructs and methods as those previously validated for disrupting conventional kinesin i 0.8 function in other cells (Verhey et al., 2001), we observed profound inhibition of insulin-stimulated GLUT4 trans­ 0 . 6 a,. location in 3T3-Ll adipocytes (Figures 7 and 8) and GLUT4-YFP vesicle movements in live cells (Figure 6C). 4 0 . Although it is not known which isotypes of kinesin light o.z. chain interact with KIF5B heavy chains in adipocytes, the TPR6 domain and the heterodimerization region of the 0.0 heptad repeats are highly conserved among the various KLC l 176 TPR6 (·) KLC L17 6 TPR6 (-) kinesin isoforms, and no preference of binding is observed Basa l Ins ulin between kinesin heavy chain and different light chain isotypes (Rahman et al., 1998). Therefore, the dominant­ 10 0 negative mutants constructed from kinesin light chain en KLCl-C used in this study are able to block efficiently 80 ·c conventional kinesin-mediated cargo transport. Taken GO together, our data are consistent with the hypothesis that KIF5B drives GLUT4 movements along microtubules as a required step in insulin's ability to stimulate GLUT4 translocation to the plasma membrane. !!! • 20 "I§:. Effect of microtubu/e disrupting agents on GLUT4 translocation KLC L 176 TPR6 N KLC L176 TPR6 (-) The data presented here raise an interesting paradox. If Basal lnsulln long-range GLUT4 movements on microtubules are required for transit and fusion of GLUT4-containing Fig. 8. Dominant-negative kinesin light chain mutants block insulin­ stimulated GLUT4 translocation to the cell surface. (A) The cell­ membranes with the plasma membrane, why is the surface anti-Myc contents of basal and insulin-treated Myc-GLUT4- complete disruption of microtubules by agents such as EGFP-transfected adipocytes (-) and the double-transfected adipocytes colchicine only partially effective in inhibiting GLUT4 (KLC, LI 76 and TPR6) were quantified. The bars represent the mean translocation? One explanation for this may be related to of the Myc/EGFP signal ratio of 20 cells for each condition. *P < the change in GLUT4 localization observed in response to 0.0001. (B) The percentage of cells displaying the cell-surface Myc rim of basal and insulin-treated single-and double-transfected 3T3-Ll disruption of rnicrotubules. Dispersion of perinuclear adipocytes (50 cells each). GLUT4 towards the cell periphery is quite dramatic under these conditions, even in the absence of insulin, reflecting the inhibition of dynein-mediated transport of GLUT4-containing vesicles towards the minus ends of is similar to models of synaptic vesicle regulation by microtubules (Emoto et al., 2001; Patki et al., 2001). Thus, neurotransmitters (Terada and Hirokawa, 2000). Our depolymerization of microtubules actually mimics the experiments designed to disrupt KIF5B function are action of insulin to relocate perinuclear GLUT4-contain­ performed over 24 h to allow for adequate expression of ing membranes throughout the cell and towards the actin­ the dominant inhibitory constructs. Thus it is not possible rich cell cortex. Insulin appears to mediate this effect to determine whether the requirements for kinesin function through kinesin-directed GLUT4 movements on micro­ on GLUT4 trafficking is acute or long term. Future tubules, while colchicine mediates the effect through experiments are needed to address this issue. blocking the return of GLUT4-containing vesicles to the perinuclear region, and perhaps by disrupting their reten­ Role of P/3-kinase in GLUT4 trans/ocation Glucose transport regulation by insulin is completely tion in this region. Another explanation for the paradoxical incomplete dependent on functional p85/p 110-type PI3-kinase (Okada inhibition of GLUT4 translocation by microtubule depoly­ et al., 1994). It was therefore surprising to observe that merizing agents may be that the acute action of insulin on microtubule-based movements of GLUT4 are apparently GLUT4 translocation is only partially dependent on totally wortmannin insensitive (Figure 3). This remarkable GLUT4 movements on microtubules. Insulin may act at finding raises two important questions. First, the data several steps in the GLUT4 trafficking pathway, each of suggest the possibility that the PI3-kinase-dependent step which can elicit an incremental increase in the overall rate or steps in the GLUT4 trafficking pathway are late steps, of exocytosis. It is also possible that microtubule-based perhaps related to the fusion of GLUT4-containing GLUT4 movements are mostly involved in replenishing membranes with the plasma membrane. This is also GLUT4-containing vesicles near the cell periphery that suggested by a report showing that synip is released from have undergone regulation by insulin. According to this syntaxin-4 upon insulin stimulation (Min et al., 1999). model, the initial acute action of insulin is targeted to This regulation of syntaxin-4, which in tum is thought to GLUT4-containing vesicles already near the cell peri­ mediate membrane fusion at the plasma membrane, is phery, and then the relatively slow long-range movements wortmannin sensitive. Secondly, our data raise the ques­ from the perinuclear region increase the GLUT4-contain­ tion of the identity of the other pathway(s) involved in ing membranes in this insulin-sensitive pool. This concept mediating insulin's effect on kinesin-directed movements 239 6 KIF5B mediates GLUT4 movements on microtubules Imaging of live adipocytes of GLUT4. It would seem that the possibility of insulin 3T3-Ll adipocytes, expressing appropriate cDNA constructs, were Ras signaling through p21 is ruled out, given that a seeded in glass-bottomed dishes (MatTek Corporation). During the Ras dominant inhibitory mutant of p21 is ineffective in treatment and recording, cells were kept in Krebs-Ringer/HEPES buffer modulating GLUT4 translocation (Hausdorff et al., 1994; (pH 7.4) supplemented with 2% BSA and 0.22 mg/ml sodium pyruvate. Throughout the 15 min time lapse, images were taken every 5 s using an Haruta et al., 1995), as is inhibition of the MEK protein Ras Olympus IX-70 inverted microscope with CCD camera. Deconvolution kinase downstream of p21 (Usui et al., 1999). This and image reconstruction of the image stacks was performed using question related to the molecular elements involved in Metamorph software (Universal Imaging). The movements of insulin signaling to modulate GLUT4 movements on GLUT4-YFP vesicles over 2 µm for three consecutive frames (10 s) microtubules will be important to address in future studies. were counted blindly and categorized into inward (toward the nucleus and/or MTOC), outward (away from the nucleus and/or MTOC) or parallel (undistinguishable) movements. KIF58 receptors on GL UT4 vesicles Affyme trix GeneChip analysis Another related issue is the mechanism whereby conven­ Expression analysis was carried out as suggested by the manufacturer tional kinesin KIF5B engages GLUT4-containing vesicles. (Affymetrix). Briefly, from three different sets of 3T3-Ll adipocytes, Recent work has identified potential receptors for the TPR mRNA was isolated using Oligotex mRNA kit (Qiagen). Double-stranded cDNA was synthesized from 5 µg mRNA, purified and biotinylated. The domains of kinesin light chains, including IlPl (Verhey cDNA was hybridized to the murine genome U74 arrays (A and B) for et al., 2001), Sunday Driver (SYD or IlP3) (Bowman et al., 16 h at 45 °C. The arrays were stained with streptavidin phycoerythrin 2000) and amyloid precursor protein (APP) (Kamal et al., solution and scanned in an HP GeneArray scanner. Average signal was 2001). Furthermore, the interaction between kinesin and calculated by subtracting signals from 11-20 overlapping 25mer probes cargo can be regulated with other proteins, such as heat by those from corresponding mismatch probes. shock chaperone 70 (Tsai et al., 2000). Also, recent work lmmunoprecipitation, mass spectrometry and western has implicated the protein kinase GSK3 as a regulator of blotting kinesin light chain interaction with membranes (Morfini Immunoprecipitation and mass spectrometry were performed as et al., 2002). These findings suggest a mechanism whereby described previously (Park et al., 2001) with 1 mg total protein and 4 µg antibodies. For western blotting, cell lysates were collected in SDS phosphorylation of kinesin light chain by GSK3 disrupts lysis buffer (20 mM HEPES, pH 7 .2, 1 % sodium dodecyl sulfate, 1 mM its interaction with receptors on membrane organelles. The sodium vanadate) supplemented with protease inhibitors and loaded onto role of phosphorylation in regulating kinesin function was 10% polyacrylarnide gels. also suggested by others (Lee and Hollenbeck, 1995; lmmunofluorescence microscopy De Vos et al., 2000; Morfini et al., 2001). This model, 3T3-Ll adipocytes were fixed with 4% formaldehyde in phosphate­ coupled with our data presented here, suggest there may be buffered saline (PBS), permeabilized and blocked with 0.5% Triton one or more receptors on GLUT4-containing vesicles that X-100 and 1 % fetal bovine serum in PBS for 20 min. Cells were bind the kinesin light chain TPR domain and are poten­ incubated with primary antibodies for 2 h and with rhodarnine- or FITC­ tially regulated by insulin signaling. Future experiments in conjugated secondary antibodies for 30 min. To analyze Myc-GLUT4- EGFP translocation in adipocytes, cells were immunostained as described our laboratory are directed to test this hypothesis. previously (Jiang et al., 2002). Briefly, the cell-surface Myc-GLUT4- GFP was visualized with anti-Myc antibody and rhodamine-labeled anti­ mouse secondary antibody without permeabilization. Where applicable, the adipocytes were then fixed, permeabilized and immunostained for Materials and methods HA-tagged proteins, using polyclonal anti-HA antibody and Alexa-350- conjugated secondary antibody. Images were taken with an Olympus IX- 70 microscope with CCD camera and then processed using Metamorph Materials software. The specific plasma membrane content of Myc-GLUT4-GFP Plasmids encoding HA-tagged rat KLCI-C and its truncated constructs, was measured as described previously (Jiang et al., 2002). For F-actin and HA-KLC-176 and HA-KLC-TPR6, were kindly provided by Dr mitochondrial staining, cells were immunostained with anti-HA antibody K.J.Verhey (Harvard Medical School). Myc-GLUT4-EGFP construct and FITC-conjugated secondary antibody. Rhodarnine-phalloidin was was prepared as described previously (Jiang et al., 2002). GLUT4-YFP added together with the secondary antibody. Mitotracker (Molecular was constructed by inserting Myc-GLUT4 into pBluescript containing a Probes) was added before initial fixation. linker with unique sites (Nhel and Agel), and then by inserting it into the same sites of pECFP-Cl vector (Clontech). For the tubulin--CFP Supplementary data construct, rat tubulin cDNA (DDBJ/EMBUGenBank accession No. Supplementary data are available at The EMBO Journal Online. NM_0ll6 55) was isolated and amplified from total RNA using standard RT-PCR. Anti-kinesin heavy chain (H2) monoclonal (Chemicon International) and anti-Myc (clone 9El0) monoclonal (Santa Cruz Biotechnology) antibodies were used. Rabbit polyclonal anti-GLUT4 Acknowledgements antibody and anti-HA polyclonal antibodies were produced as described This work was supported by grants from the National Institutes of Health previously (Langille et al., 1999). The Alexa 350-conjugated anti-rabbit (DK 30898 and DK 60837) to M.P.C. and a Postdoctoral Fellowship from antibody was from Molecular Probes. Human insulin was obtained from the American Diabetes Association to S.S. Eli Lilly Co. All other chemicals were from Sigma unless otherwise stated. References Cell culture, transfection and treatment 3T3-Ll fibroblasts were cultured and differentiated into adipocytes as Abel,E.D., Peroni,O., Kim,J.K., Kim,Y.B., Boss,O., Hadro,E., described previously (Park et al., 2001). Four days after starting Minnemann,T., Shulman,G.I. and Kahn,B.B. 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Conventional kinesin KIF5B mediates insulin‐stimulated GLUT4 movements on microtubules

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Abstract

The EMBO Journal Vol. 22 No.10 pp. 2387-2399, 2003 Conventional kinesin KIFSB mediates insulin­ stimulated GLUT4 movements on microtubules mechanism whereby insulin signaling causes intracellular Sabina Semiz, Jin G.Park, membranes containing GLUT4 to move and fuse with the Sarah M.C.Nicoloro, Paul Furcinitti, plasma membrane requires the p85/p 110 type phos­ Chuanyou Zhang, Anil Chawla, John Leszyk phatidy linositol 3 (PI3)-kinase (Cheatham et al., 1994; and Michael P.Czech Okada et al., 1994; Sharma et al., 1998) and appears to Program in Molecular Medicine, 373 Plantation Street, University of involve the downstream protein kinase Akt2 (Hill et al., Massachusetts Medical School, Worcester, MA 01605, USA 1999; Cho et al., 2001). Some components involved in Corresponding author membrane trafficking of GLUT4 have also been identified, e-mail: [email protected] including the SNARE proteins V AMP2 (Cheatham et al., 1996; Martin et al., 1996) and syntaxin-4 (Volchuk et al., S.Semiz and J.G.Park contributed equally to this work 1996; Olson et al., 1997; Pessin et al., 1999), as well as SNAP25 (Jagadish et al., 1996) and SNAP23 (Kawanishi Insulin stimulates glucose uptake in muscle and adi­ et al., 2000). In both primary and cultured adipocytes, pose cells by mobilizing intracellular membrane vesicles containing GLUT4 glucose transporter GLUT4 recycles slowly between intracellular membranes proteins to the plasma membrane. Here we show in and the plasma membrane even in the absence of insulin, live cultured adipocytes that intracellular membranes and recycling is markedly enhanced by activation of containing GLUT4-yellow fluorescent protein (YFP) GLUT4 exocytosis by the hormone (Jhun et al., 1992; move along tubulin--cyan fluorescent protein-labeled Yang and Holman, 1993). A PI3-kinase-dependent step in microtubules in response to insulin by a mechanism this process may be fusion of GLUT4-containing mem­ that is insensitive to the phosphatidylinositol 3 (PI3)­ branes with the plasma membrane, based on the identifi­ kinase inhibitor wortmannin. Insulin increased by cation of the insulin-regulated protein synip as an apparent several fold the observed frequencies, but not veloci­ modulator of syntaxin-4 function (Min et al., 1999). ties, of long-range movements of GLUT4-YFP on Evidence has been steadily accumulating in favor of the microtubules, both away from and towards the peri­ hypothesis that GLUT4 movements in adipocytes and nuclear region. Genomics screens show conventional muscle cells are also dependent on cytoskeletal structures. kinesin KIFSB is highly expressed in adipocytes and Many laboratories have confirmed that optimal GLUT4 this kinesin is partially co-localized with perinuclear translocation in response to insulin requires intact actin GLUT4. Dominant-negative mutants of conventional filaments (Wang et al., 1998; Omata et al., 2000; Bose kinesin light chain blocked outward GLUT4 vesicle et al., 2001; Emoto et al., 2001; Patki et al., 2001; Jiang movements and translocation of exofacial Myc-tagged et al., 2002), and co-localization of GLUT4 and GLUT4-green fluorescent protein to the plasma mem­ polymerized actin is observed in insulin-treated muscle brane in response to insulin. These data reveal that cells (Asahi et al., 1999; Khayat et al., 2000) and insulin signaling targets the engagement or initiates adipocytes (unpublished data). We found that depolymer­ the movement of GLUT4-containing membranes on ization of intermediate filaments and microtubules also microtubules via conventional kinesin through a PI3- disrupted perinuclear GLUT4 localization, as well impair­ kinase-independent mechanism. This insulin signaling ing GLUT4 responsiveness to insulin (Guilherme et al., pathway regulating KIFSB function appears to be 2000; Emoto et al., 2001). Numerous laboratories have required for GLUT4 translocation to the plasma confirmed that depolymerization of microtubules partially membrane. inhibits GLUT4 recycling and insulin-mediated GLUT4 Keywords: adipocytes/GLUT4/insulin/kinesin/ translocation (Fletcher et al., 2000; Emoto et al., 2001; microtubules Molero et al., 2001; Olson et al., 2001). Live cell imaging of 3T3-Ll adipocytes expressing GLUT4-green fluores­ cent protein (GFP) has revealed short sporadic GLUT4 movements, as well as longer range movements, that are Introduction disrupted by colchicine (Fletcher et al., 2000). However, Glucose homeostasis in humans is dependent upon the while there is agreement that endocytosis of GLUT4 to catalytic activity of a glucose transporter protein, GLUT4, perinuclear membranes is driven by dynein motors on that is highly expressed in fat and muscle and responds to microtubule tracks, a recent report has questioned the insulin by translocation from intracellular vesicles to the involvement of microtubules and kinesins in the outward plasma membrane (Olson and Pessin, 1996; Czech and process (Molero et al., 2001). Another report concludes Corvera, 1999; Bryant et al., 2002). Ablation of GLUT4 outright that microtubules are not required for GLUT4 expression in either adipocytes (Abel et al., 2001) or translocation (Shigematsu et al., 2002). Thus, the issue of skeletal muscle (Zisman et al., 2000) of mice can lead to microtubule and kinesin involvement in insulin-mediated glucose intolerance, insulin resistance and diabetes. The GLUT4 movements has remained unresolved. © European Molecular Biology Organization 2387 S.Semiz et al. The aim of this study was to visualize directly GLUT4- suggest that an individual GLUT4-YFP-containing yellow fluorescent protein (YFP) movements in live vesicle may be bound to and carried by both minus end­ insulin-sensitive 3T3-Ll adipocytes under conditions and plus end-directed motor proteins. where microtubules could also be observed. Here we report images showing fluorescence from GLUT4-YFP Insulin signaling increases the frequency of moving along microtubules labeled with tubulin--cyan long-range GLUT4-YFP movements fluorescent protein (CFP), providing direct evidence that Experiments were conducted to determine whether insulin membranes containing GLUT4 move on microtubule action modulates long-range movements of GLUT4-YFP­ tracks. Remarkably, insulin treatment is shown to increase containing vesicles on microtubules. Analysis of numer­ the frequency of these long-range movements both away ous movies of control versus insulin-treated 3T3-Ll from and towards the perinuclear region. The outward adipocytes expressing both GLUT4-YFP and tubulin­ movements of GLUT4 towards the plus ends of micro­ CFP revealed no significant effect of the hormone on the tubules appear to be physiologically relevant because mean velocities of GLUT4-YFP movements in either these movements as well as insulin-mediated GLUT4 inward or outward directions (Figure 3A). There was also translocation to the plasma membrane was blocked by no consistent change in the distribution of velocities dominant inhibitory kinesin light chain proteins. These exhibited by various groups of GLUT4-YFP-containing new data are consistent with the hypothesis that GLUT4- vesicles (Figure 3B). However, insulin signaling in these containing membranes are cargo for conventional kinesin, cultured adipocytes caused a marked increase in the which moves these membranes towards the cell periphery observed frequency of total long-range movements of in response to insulin. GLUT4-YFP on microtubules (Figure 3C). This increase in the number of such events per unit time was highly significant when either inward ( ~ 3-fold increase) or Results outward ( ~8-fold increase) directions were monitored. GLUT4-containing membranes move on Thus insulin-treated adipocytes display much higher numbers of GLUT4-YFP-containing vesicles engaged in microtubules Preliminary imaging performed on live 3T3-Ll adipocytes extended linear motion along microtubules than untreated expressing GLUT4-GFP or GLUT4-YFP confirmed two cells at any given time. earlier reports that showed very active movements of To further confirm that these long-range movements fluorescent vesicles which were mostly randomly directed represent microtubule-based GLUT4-YFP transport, and short range (Oatey et al., 1997; Patki et al., 2001). microtubules were depolymerized by treatment with Occasional linear movements over distances of 10 µm or colchicine. As expected, the colchicine treatment resulted more could be observed as well, as noted previously in the loss of our ability to visualize microtubules with (Oatey et al., 1997). To examine whether these long-range tubulin-CFP (unpublished data) and the abolition of movements are microtubule based, 3T3-Ll adipocytes insulin-stimulated long-range GLUT4-YFP movements electroporated with plasmids encoding both GLUT4-YFP (Figure 3C). Interestingly, very active short-range random and tubulin--CFP were examined by time-lapse micro­ movements of GLUT4-YFP-containing vesicles could scopy at a single optical plane. Under these conditions, still be observed in the periphery of the colchicine-treated complex networks of labeled microtubules could be cells, which may represent movements on other cyto­ visualized throughout the cultured adipocytes (Figure 1). skeletal structures, such as F-actin. These results demon­ Remarkably, the majority of the long-range GLUT4-YFP­ strate the specific requirement of intact microtubules for containing vesicle movements occurred along the micro­ long-range movements of GLUT4-YFP in cultured tubule tracks within this network (Figures 1 and 2). adipocytes in response to insulin. Figure 1 shows a typical single long-range movement of GLUT4-YFP fluorescence over a distance of ~10 µm in Insulin signaling to increase long-range 40 s. Moreover, some vesicles could be observed to travel movements of GLUT4-YFP on microtubules is over a total distance of 20 µm by transiting onto several Pl3-kinase independent different microtubules. These data directly demonstrate It is likely that there are multiple steps in the regulated that the long-range movements of GLUT4-YFP vesicles movements and fusion of GLUT4-containing membranes are associated with microtubule-based motor activity with the plasma membrane. These steps might include rather than being random, Brownian or based on other membrane budding or release of membranes from tether­ cytoskeletal components. ing in the perinuclear region, movement along micro­ Using the microtubule organizing center (MTOC) and/ tubules (Figures 1-3), movement along actin filaments or the nuclei as visual indicators, we were able to (Omata et al., 2000; Bose et al., 2001; Jiang et al., 2002), distinguish the directionality of many of the GLUT4- and docking and fusion with the plasma membrane YFP movements. Figure 2 shows three examples in which (Cheatham et al., 1996; Martin et al., 1996; Volchuk the identical GLUT4-YFP-containing vesicles were cap­ et al., 1996; Min et al., 1999). Insulin regulation of this able of moving both towards (right panels) and away from overall process resulting in increased GLUT4 proteins in (left panels) the perinuclear region of the adipocyte. These the plasma membrane and enhanced glucose transport into directions are referred to as 'inward' and 'outward', adipocytes is totally inhibited by agents that disrupt PI3- respectively, in Figure 2. Analysis of the velocities of kinase (Cheatham et al., 1994; Okada et al., 1994; Sharma these long-range movements in many cells (Figures 2 and et al., 1998). This could mean that: (i) all the above 3A) showed that the speeds of outward and inward regulated steps depend on PI3-kinase; (ii) as few as one movements were not significantly different. These data step in the pathway exhibits such dependency; or (iii) none 2388 KIF5B mediates GLUT4 movements on microtubules Fig. 1. GLUT4-YFP vesicles move on microtubules in cultured adipocytes (a time-lapse movie is presented in the Supplementary data available at The EMBO Journal Online). (A) 3T3-Ll adipocytes were electroporated with plasmids encoding GLUT4-YFP and tubulin-CFP. After serum starva­ tion, cells were treated with 100 nM insulin for 15 min, followed by time-lapse image recording with a 5 s interval up to 15 min. Shown here is a merged image of the first and the last frames of a 40 s sequence, showing that a GLUT4-YFP-containing vesicle (red) adjacent to microtubule (green) moves from right to left as indicated by arrows with time points (seconds). N, nucleus. (B) Nine sequential frames of boxed area in (A) are enlarged and merged. Positions of GLUT4-YFP vesicles are indicated by arrows with elapsed time (seconds). of the regulated steps are PI3-kinase dependent, but part of (Figure 3A) or the distribution of the velocities among the machinery of forming the regulatable sequestration vesicle populations (Figure 3B). compartment or other aspect of the cell machinery Based on this failure of wortmannin to inhibit linear necessary for the response is PI3-kinase dependent. In GLUT4-YFP movements, we wanted to confirm the order to examine whether insulin regulates linear GLUT4- known effect of the drug to inhibit GLUT4 translocation YFP movements on rnicrotubules through a PI3-kinase­ to the plasma membrane in response to insulin under these dependent mechanism, we treated 3T3-Ll adipocytes with same experimental conditions (Figure 4). Using a wortmannin prior to addition of insulin. Under the GLUT4-GFP construct with a Myc tag on the first conditions of these experiments wortmannin abolished exofacial loop as reporter, it was found that the insulin­ the strong increase in phosphorylation of the protein stimulated increase in cell-surface Myc-GLUT4-GFP was kinase Akt in response to insulin (data not shown). indeed nearly, but not completely, inhibited (74%) by Surprisingly, this treatment did not affect the number of 100 nM wortmannin, similar to a previous report long-range GLUT4-YFP movements per unit time in (Hausdorff et al., 1999). Interestingly, however, 46% of insulin-treated cells (Figure 3C), the average maximal these wortmannin- and insulin-treated cells did display a velocities in either inward or outward GLUT4 movements strong rim of GLUT4-GFP fluorescence around the cell 2389 S.Semiz et al. Inward Outward 0.328 µm sec· 1 0.346 µm sec· 1 0.160 µm sec·1 0.392 µm sec· 1 0.164 µm sec· 0.272 µm sec· 1 Fig. 2. A single GLUT4-YFP vesicle moves bi-directionally on microtubules. Three different GLUT4-YFP vesicles from time-lapse images that displayed bi-directional movements are shown. The movements were categorized as 'inward' and 'outward', as described in the text. The numbers inside images indicate elapsed time (seconds) from the first frame of each sequence. Right and left panels show bi-directional movements of the identical vesicles in the same field of cells. The numbers below images indicate maximal velocity of vesicles. Bars, 4 µm. periphery (Figure 4B). These data are consistent with the hypothesis suggests that the fusion step whereby Myc­ hypothesis that insulin acts to move GLUT4-containing GLUT4-GFP is inserted into the plasma membrane to vesicles towards the cell surface on rnicrotubules in the become accessible to Myc antibody in unpermeabilized cells requires PB-kinase. presence of wortmannin. According to this model, GLUT4-containing vesicles in cells treated with insulin plus wortmannin can then dock with the plasma mem­ Insulin-stimulated GLUT4 translocation requires brane, leading to the rim of Myc-GLUT4-GFP at the sub­ conventional kinesin KIF58 plasma membrane region observed by monitoring the GFP Plus end-directed transport of vesicles and organelles on signal but not the exofacial Myc signal (Figure 4A). Some rnicrotubules is mediated by the large family of kinesin of these vesicles are perhaps returned to the perinuclear motor proteins. In a screen for genes highly expressed in region, while others accumulate as docked vesicles. This 3T3-Ll adipocytes using Affymetrix GeneChip arrays 2390 KIF5B mediates GLUT4 movements on microtubules GFP aMyc A A 0.6 ..__ Insulin □ Basal - - � - ■ Insulin ■ Wort + Insul in - - - - f-- - -- -- Inward Outward Wort+ Insulin �4 □ Basal +- -- �1-- --i ■ Insulin -- ------- --- �3 ■ Wort + Insulin � 2 G1 !o 0,2 0.4 0.6 M 0.2 0. 0.6 0.8 .. Inward velocity Outward velocity 1 1 (µm sec· ) f ,m aec· ) c:: 1.4 • *" □ Basal nr::7 1.2 - - -+ -- -- - --1 ■ Insulin Wort+ Insulin ----- 1.0 1-- --1 l2l Col + Insulin 1ia 0B 0.6 GI 0 , 4 0.2 "" 0.0 Fig. 4. Accumulation of GLUT- YFP-containing vesicles near the cell Total Inward Outward periphery in adipocytes treated with wortrnannin plus insulin. (A) 3T3- Ll cells were electroporated with the Myc-GLUT4-EGFP plasmid, starved, and then either treated with wortrnannin for 15 min and then Fig. 3. Insulin increases the number, but not velocities, of long-range 100 nM insulin for 30 min (Wort + Insulin), or 100 nM insulin alone GLUT4-YFP vesicle movements on microtubules in a wortmannin­ for 30 min (Insulin). Cells were then fixed and stained for plasma mem­ insensitive manner. 3T3-Ll adipocytes expressing GLUT4-YFP and brane GLUT4 (red) with anti-Myc antibody and rhodamine-conjugated tubulin-CFP were starved and either left untreated (Basal), or treated secondary antibody without permeabilization. EGFP signal is shown in with either 100 nM insulin (Insulin), 100 nM wortrnannin for 15 min green. Bar, 10 µm. (B) Myc-GLUT4-EGFP-electroporated 3T3-Ll followed by a 15 min insulin treatment (Wort + Insulin), or 50 µM adipocytes were starved and left untreated (Basal), treated with either colchicine for 2 h followed by a 15 min insulin treatment (Col + 100 nM wortmannin for 45 min (Wort) or 100 nM insulin for 30 min Insulin). Long-range movements of GLUT4-YFP vesicles from 6 to 16 (Insulin), or pre-treated with 100 nM wortrnannin for 15 min followed time-lapse images for each condition were analyzed for their velocities by a 30 min 100 nM insulin treatment (Wort + Ins). For each set, 50 and number of events. (A) Average maximal velocities of GLUT4- cells were counted for the EGFP and the Myc rims. YFP vesicles moving either towards (inward) or away from (outward) the perinuclear region. Total vesicle counts represent the sum of all long-range GLUT4-YFP vesicle movements observed, including those where directionality could not be unequivocally determined. Error bars adipocytes compared with fibroblasts was observed by represent the SEM. (B) Distribution of maximal velocities of GLUT4-YFP vesicles. (C) The number of GLUT4-YFP vesicle move­ western blot analysis (Figure 5A). Conventional kinesin ments in a unit area is shown. Error bars represent the SEM. *P = KIF5B is composed of two heavy chains and two light 0.0006; **P = 0.002; #P = 0.01; ##P = 0.004. chains in a heterotetrameric configuration (Figure 6A) and is expressed in many tissues, while the other conventional kinesin isotypes KIF5A and KIF5C function mainly in (U74 series), oligonucleotides derived from the gene neuronal tissues (Goldstein and Yang, 2000). Since the sequence of the conventional kinesin heavy chain KIF5B anti-KIF5 antibody (H2) we used recognizes all three were found to yield the highest signal of all the kinesins isotypes but has lowest affinity for KIF5B (Kanai et al., represented on the arrays (Table I). Furthermore, higher 2000; Cai et al., 2001), we performed immunoprecipit­ expression of conventional heavy chain in cultured ation with the H2 antibody followed by mass spectrometry 239 1 439 S.Semiz et al. meric kinesin complex and block the kinesin--cargo Table I. Expression of kinesins in 3T3-Ll adipocytes was analyzed binding. Both the HA-KLC-L176 and HA-KLC-TPR6 by Affymetrix GeneChips constructs have been shown to disrupt conventional Kinesin type Average SEM Chip Probe set kinesin function when expressed in other cell types signal set (Verhey et al., 2001). We also employed expression of KIF5B 20 732 2220 B 113588_f_at the native kinesin light chain in these experiments as a KIF21B 4063 666 B 11482l_at control. After electroporation with these HA- or CFP­ KIF3B 3520 996 B 115208_at conjugated constructs, 3T3-Ll adipocytes remained intact KIF3A 2829 334 B 115929_at and showed no phenotypic defect. All three KLC KIF21A 204 1 572 B 11723l_at KIFlB 1579 192 B 115895_at constructs localized mostly in the perinuclear region, KIF1 3A 853 334 B 109305_at while two dominant-negative KLC mutants often mis­ KIF2 574 168 A 99962_at localized into the nucleus upon higher expression (Figures KIF3C 377 303 A 93635_at 6B, and 7 A and B). Consistent with the known function of KIF4 274 42 A 104644_at KIFC2 123 68 A 9389l_at conventional kinesin in mitochondrial dispersion (Tanaka KIFC3 73 105 A 104335_at et al., 1998), cells expressing the dominant-negative KLC KIFlA 52 119 A 92890_at mutants displayed reduced mitochondrial staining in the KIFCl -978 461 A 98471_f_at peripheral cytoplasm (Figure 6B). Confirming the speci­ KLCl 786 90 A 93565_at ficity of the dominant-negative mutant function, expres­ KLC2 242 A 102636_at sion of either the full-length or the dominant-negative KLC proteins did not affect intermediate filament distri­ Shown here are the average signal representing the mean of average bution (data not shown) or insulin-stimulated cortical actin differences and the SEM from three independent experiments. The names of murine genome chip sets (U74A and U74B) and individual rearrangement (Figure 6B). To test whether vesicle probe sets giving the highest signal for a particular kinesin are shown. movements stimulated by insulin are mediated by Expression signals of KLCl and KLC2 are also presented. KIF5B, 3T3-Ll adipocytes were electroporated with the plasmid containing CFP-conjugated KLC constructs and GLUT4-YFP, and counted for the number of vesicle in order to confirm the expression of KIF5B. As shown in movements toward the cell periphery by live cell imaging. Figure 5B and C, a single major band was detected at Figure 6C showed that the dominant-negative KLC ~120-130 kDa after an NP-40 extraction in both starved mutants, but not the full-length KLC, significantly and insulin-stimulated conditions. Mass spectrometry inhibited insulin-stimulated outward long-range move­ analysis of the band identified peptides with sequences ments of GLUT4-containing vesicles. Together with high that exactly match the murine kinesin heavy chain KIF5B, expression of KIF5B (Figure 5), these data suggest that indicating that only this isotype is present in 3T3-Ll KIF5B is required for mobilizing GLUT4-containing adipocytes (Figure 5C). Experiments were therefore vesicles in response to insulin in 3T3-Ll adipocytes. designed to test the hypothesis that conventional kinesin We next tested whether these outward GLUT4-contain­ KIF5B function is required for GLUT4 translocation to ing vesicle movements mediated by KIF5B are required the plasma membrane in response to insulin. First, the for insulin-stimulated translocation of GLUT4 to the intracellular localizations of endogenous conventional plasma membrane. As shown in Figure 7 A, expression of kinesin and GLUT4 were analyzed in 3T3-Ll adipocytes either dominant-negative mutant KLC abolished the by immunostaining. The kinesin heavy chain is mainly ability of insulin to stimulate translocation of exofacial present in the perinuclear region of 3T3-Ll adipocytes and Myc-GLUT4-GFP to the cell surface, as reflected by the partially co-localizes with GLUT4-containing vesicles absence of anti-Myc antibody binding to adipocytes under (Figure 5D). These data are consistent with the hypothesis these conditions. In contrast, intact adipocytes expressing that this kinesin is associated with intracellular GLUT4- Myc-GLUT4-GFP and the native kinesin light chain containing membranes in intact cultured adipocytes. displayed strong anti-Myc antibody binding to the cell Next we employed two dominant inhibitory kinesin surface in response to insulin (Figure 7 A and B). light chain mutant proteins, based on the KLCl isoform, to Quantitation of the results from a large number of disrupt conventional kinesin KIF5B function (Figure 6A). adipocytes in these experiments was performed by visually The KLCl isoform was found to be expressed in counting the number of cells that display cell-surface adipocytes, while KLC-2 is not (Table I). One of the binding of anti-Myc antibody (Figure 8A), as well as by constructs, hemagglutinin (HA)-tagged KLC-TPR6 (or quantifying the ratio of signal intensity from the anti-Myc CFP-KLC-TPR6), lacks the heptad repeats domain that antibody binding (cell-surface Myc-GLUT4-GFP) versus functions as the kinesin heavy chain binding region the signal from the total GFP fluorescence (total expressed (Diefenbach et al., 1998; Verhey et al., 1998; Kamal and Myc-GLUT4-GFP) (Figure 8B). These techniques for Goldstein, 2002). Its expression is predicted to block estimating the action of insulin on translocation of Myc­ interaction between endogenous kinesin and cargo GLUT4-GFP have been described in detail previously proteins by saturating kinesin receptors such as those (Park et al., 2001; Jiang et al., 2002). By both these that might be present in GLUT4-containing vesicles. The measurements, expression of the inhibitory kinesin mutant other construct, HA-tagged KLC-Ll 76 (or CFP-KLC­ proteins exerted virtually complete abolition of cell-surface Ll 76), lacks the C-terminal TPR6 domain involved in Myc-GLUT4-GFP display in response to insulin (Figures 7 KLC--cargo binding (Skoufias et al., 1994; Bi et al., 1997). and 8), consistent with a requirement of conventional This mutant is predicted to incorporate into heterotetra- kinesin for insulin action on GLUT4 translocation. 2392 KIF5B media tes GLUT4 movements on microtubule s IP : lgG H2 Ad i Fib B kDa Basal Insulin Basal lnsur in 175- 17 5 - KHC 83- 62- .-KHC 18: H2 47- 2 · lg 25- IP: H2 Ne Insulin Lysa1e Sl iver stain aKHC aG LUT4 Merg e Fig. 5. Endogenous conventional kinesin KIF5B partially co-localizes with GLUT4 in 3T3-Ll adipocytes. (A) Confluent fibroblasts (Fib) and fully diff erentiated 3T3-Ll adipocytes (Adi) were analyzed by irnrnunoblotting using anti-kinesin heavy chain H2 antibodies. (B) Starved (Basal) and insulin-stimulated 3T3-Ll adipocytes were lysed and subject to irnrnunoprecipitation (IP) with either non-immune mouse IgG (lgG) and anti-kinesin heavy chain H2 antibody. lg, immunoglobulin. (C) A gel was prepared as above and silver stained. The 120 kDa band was excised and analyzed by mass spectrometry. Shown are two peptides for which the amino acid sequences were matched to the murine conventional kinesin heavy chain KIF5B. (D) 3T3-Ll adipocytes were immunostained with anti-kinesin heavy chain H2 antibody (aKHC; red) and anti-GLUT4 antibody (aGLUT4; green). Two different cells are shown with merged images (Merge) of both channels. Bars, 10 µm. 2393 S.Semiz et al. A C c 1. 0 �---------------� (·) 1 o.e ] Globu lar ta il 8 � 0.6 +----------- Corled-coil I 0.4 - -- ii 0.2 -t- -,.....-=1=----.=!=;- -1 ... TPR motifs 0.0 KLC Ba.sal Insul in HA 176 L1 76 TPR6 Insulin KLC Basal L1 76 TPR6 aHA Actin aHA Mita Fig. 6. Dominant-negative KLC mutants inhibit insulin-stimulated GLUT4-YFP vesicle movements. (A) Top diagram shows heterotetrameric structure of conventional kinesin and a proposed model for cargo binding (MT, microtubule). Structures of wild type (KLC), and C-terminal (Ll76) and an N-terminal deletion (TPR6) mutants of kinesin light chain are shown in the bottom diagram. All constructs have HA tags within their N-termini. The numbers represent amino acid sequences. These diagrams are adapted from Verhey et al. (2001). (B) Plasmids containing the full-length (KLC) and deletion mutant (Ll 76 and TPR6) constructs were electroporated into 3T3-Ll adipocytes. Cells were starved and either left untreated (Basal) or stimulated with insulin for 30 min. Electroporated cells were identified by irnmunostaining with anti-HA antibody ( cdIA). F-actin and mitochondria (Mito) were visualized with rhodarnine-phalloidin and Mitotracker, respectively. Bar, 10 µm. (C) 3T3-Ll adipocytes were electroporated with plasmids containing GLUT4-YFP and the kinesin light chain-CFP constructs. The number of GLUT4-YFP vesicles moving away from the nucleus in either basal or insulin-stimulated cells was counted. Five to seven cells were counted for each condition. Error bars represent the SEM. *P = 0.019; **P = 0.016. ■ KLC ■ L176 0 TPR8, ....._________.� KIF5B mediates GLUT4 movem ents on microtubules aHA GFP-G LUT4 nMyc aHA aMyc KlC ·. ·• (-) L176 Basal KLC TPR6 KLC L1 76 L176 In sulin . TP R6 TPR6 Fig. 7. Dominant-negative kinesin light chain mutants block insulin-stimulated GLUT4 translocation to the cell surface. (A) 3T3-Ll cells were electro­ porated with the Myc-GLUT4-EGFP plasmid and plasmid containing either the wild-type or dominant-negative KLC constructs. Cells were starved and either left untreated (Basal) or incubated with 100 nM insulin (Insulin), and immunostained for the plasma membrane-fused GLUT4 with anti­ Myc antibody (cxMyc) without permeabilization. Cells were then permeabilized and immunostained with anti-HA antibody (o;HA). Low magnification images show single-transfected (Myc-GLUT4-EGFP) cells and double-transfected (GLUT4-Myc-EGFP and KLC constructs) cells in the same viewing fields. Bars, 20 µm. (B) High magnification images of insulin-treated 3T3-Ll adipocytes that were either single-(-) or double-transfected as described in (A). Bars, 10 µm. Discussion translocation (Guilherme et al., 2000; Emoto et al., 2001). The present findings directly establish the conclusion that GLUT4-containing membranes move on microtubules in Long-range transport of GLUT4 vesicles on an outward direction towards the periphery of cultured microtubules adipocytes, as well as in an inward direction towards the A key finding of this study is the observed movement of perinuclear region. fluorescence signal from GLUT4-YFP along tracks delineated by microtubules labeled with tubulin-CFP (Figures 1 and 2). Furthermore, our data document such KIF58 mediates GLUT4 vesicle movements on linear movements of GLUT4-YFP over distances as long microtubules as 20 µm, directed towards both the plus and minus ends of A second significant finding reported here is the identifi­ cation of conventional kinesin KIFSB as a molecular microtubules (Figures 2 and 3). Remarkably, insulin motor required for GLUT4 translocation in response to stimulates the number of these events observed per unit time in cultured adipocytes (Figure 3). Depolymerization insulin (Figures 7 and 8). Conventional kinesin was first of microtubules by colchicine results in complete inhib­ identified as a processive molecular motor involved in ition of long-range GLUT4-YFP-containing vesicle driving vesicles and organelles in axons of vertebrate and movements (Figure 3C). Only a relatively small number squid brain (Brady, 1985; Vale et al., 1985). Kinesin heavy of GLUT4 vesicles can be seen traveling over long chain has a microtubule plus end-directed motor domain, distances after insulin stimulation relative to the total while kinesin light chain is generally thought to be number of GLUT4-containing membranes observed in a involved in kinesin binding to cargos, mediating their single field. However, our live cell microscopy is motility toward the cell periphery (Hirokawa, 1993; performed at a single optical plane, which probably Skoufias and Scholey, 1993). Recent reports demonstrated provides an underestimate of the total events per cell. that conventional kinesin is essential for mitochondrial These data extend previous indirect evidence suggesting (Tanaka et al., 1998), lysosomal (Tanaka et al., 1998) and that GLUT4-containing membranes are mobile on micro­ endosomal (Nielsen et al., 1999) transport, as well as for tubules based on the distance and linearity of their the microtubule-based motility of virus particles in movements (Oatey et al., 1997) as well as on the inhibitory infected cells (Rietdorf et al., 2001). Of the three isotypes actions of microtubule depolymerizing agents on GLUT4 of conventional kinesin known, only KIF5B could be 239 5 S.Semiz et al . detected in adipocytes. Using the same dominant inhibi­ 1. 0 tory kinesin light chain constructs and methods as those previously validated for disrupting conventional kinesin i 0.8 function in other cells (Verhey et al., 2001), we observed profound inhibition of insulin-stimulated GLUT4 trans­ 0 . 6 a,. location in 3T3-Ll adipocytes (Figures 7 and 8) and GLUT4-YFP vesicle movements in live cells (Figure 6C). 4 0 . Although it is not known which isotypes of kinesin light o.z. chain interact with KIF5B heavy chains in adipocytes, the TPR6 domain and the heterodimerization region of the 0.0 heptad repeats are highly conserved among the various KLC l 176 TPR6 (·) KLC L17 6 TPR6 (-) kinesin isoforms, and no preference of binding is observed Basa l Ins ulin between kinesin heavy chain and different light chain isotypes (Rahman et al., 1998). Therefore, the dominant­ 10 0 negative mutants constructed from kinesin light chain en KLCl-C used in this study are able to block efficiently 80 ·c conventional kinesin-mediated cargo transport. Taken GO together, our data are consistent with the hypothesis that KIF5B drives GLUT4 movements along microtubules as a required step in insulin's ability to stimulate GLUT4 translocation to the plasma membrane. !!! • 20 "I§:. Effect of microtubu/e disrupting agents on GLUT4 translocation KLC L 176 TPR6 N KLC L176 TPR6 (-) The data presented here raise an interesting paradox. If Basal lnsulln long-range GLUT4 movements on microtubules are required for transit and fusion of GLUT4-containing Fig. 8. Dominant-negative kinesin light chain mutants block insulin­ stimulated GLUT4 translocation to the cell surface. (A) The cell­ membranes with the plasma membrane, why is the surface anti-Myc contents of basal and insulin-treated Myc-GLUT4- complete disruption of microtubules by agents such as EGFP-transfected adipocytes (-) and the double-transfected adipocytes colchicine only partially effective in inhibiting GLUT4 (KLC, LI 76 and TPR6) were quantified. The bars represent the mean translocation? One explanation for this may be related to of the Myc/EGFP signal ratio of 20 cells for each condition. *P < the change in GLUT4 localization observed in response to 0.0001. (B) The percentage of cells displaying the cell-surface Myc rim of basal and insulin-treated single-and double-transfected 3T3-Ll disruption of rnicrotubules. Dispersion of perinuclear adipocytes (50 cells each). GLUT4 towards the cell periphery is quite dramatic under these conditions, even in the absence of insulin, reflecting the inhibition of dynein-mediated transport of GLUT4-containing vesicles towards the minus ends of is similar to models of synaptic vesicle regulation by microtubules (Emoto et al., 2001; Patki et al., 2001). Thus, neurotransmitters (Terada and Hirokawa, 2000). Our depolymerization of microtubules actually mimics the experiments designed to disrupt KIF5B function are action of insulin to relocate perinuclear GLUT4-contain­ performed over 24 h to allow for adequate expression of ing membranes throughout the cell and towards the actin­ the dominant inhibitory constructs. Thus it is not possible rich cell cortex. Insulin appears to mediate this effect to determine whether the requirements for kinesin function through kinesin-directed GLUT4 movements on micro­ on GLUT4 trafficking is acute or long term. Future tubules, while colchicine mediates the effect through experiments are needed to address this issue. blocking the return of GLUT4-containing vesicles to the perinuclear region, and perhaps by disrupting their reten­ Role of P/3-kinase in GLUT4 trans/ocation Glucose transport regulation by insulin is completely tion in this region. Another explanation for the paradoxical incomplete dependent on functional p85/p 110-type PI3-kinase (Okada inhibition of GLUT4 translocation by microtubule depoly­ et al., 1994). It was therefore surprising to observe that merizing agents may be that the acute action of insulin on microtubule-based movements of GLUT4 are apparently GLUT4 translocation is only partially dependent on totally wortmannin insensitive (Figure 3). This remarkable GLUT4 movements on microtubules. Insulin may act at finding raises two important questions. First, the data several steps in the GLUT4 trafficking pathway, each of suggest the possibility that the PI3-kinase-dependent step which can elicit an incremental increase in the overall rate or steps in the GLUT4 trafficking pathway are late steps, of exocytosis. It is also possible that microtubule-based perhaps related to the fusion of GLUT4-containing GLUT4 movements are mostly involved in replenishing membranes with the plasma membrane. This is also GLUT4-containing vesicles near the cell periphery that suggested by a report showing that synip is released from have undergone regulation by insulin. According to this syntaxin-4 upon insulin stimulation (Min et al., 1999). model, the initial acute action of insulin is targeted to This regulation of syntaxin-4, which in tum is thought to GLUT4-containing vesicles already near the cell peri­ mediate membrane fusion at the plasma membrane, is phery, and then the relatively slow long-range movements wortmannin sensitive. Secondly, our data raise the ques­ from the perinuclear region increase the GLUT4-contain­ tion of the identity of the other pathway(s) involved in ing membranes in this insulin-sensitive pool. This concept mediating insulin's effect on kinesin-directed movements 239 6 KIF5B mediates GLUT4 movements on microtubules Imaging of live adipocytes of GLUT4. It would seem that the possibility of insulin 3T3-Ll adipocytes, expressing appropriate cDNA constructs, were Ras signaling through p21 is ruled out, given that a seeded in glass-bottomed dishes (MatTek Corporation). During the Ras dominant inhibitory mutant of p21 is ineffective in treatment and recording, cells were kept in Krebs-Ringer/HEPES buffer modulating GLUT4 translocation (Hausdorff et al., 1994; (pH 7.4) supplemented with 2% BSA and 0.22 mg/ml sodium pyruvate. Throughout the 15 min time lapse, images were taken every 5 s using an Haruta et al., 1995), as is inhibition of the MEK protein Ras Olympus IX-70 inverted microscope with CCD camera. Deconvolution kinase downstream of p21 (Usui et al., 1999). This and image reconstruction of the image stacks was performed using question related to the molecular elements involved in Metamorph software (Universal Imaging). The movements of insulin signaling to modulate GLUT4 movements on GLUT4-YFP vesicles over 2 µm for three consecutive frames (10 s) microtubules will be important to address in future studies. were counted blindly and categorized into inward (toward the nucleus and/or MTOC), outward (away from the nucleus and/or MTOC) or parallel (undistinguishable) movements. KIF58 receptors on GL UT4 vesicles Affyme trix GeneChip analysis Another related issue is the mechanism whereby conven­ Expression analysis was carried out as suggested by the manufacturer tional kinesin KIF5B engages GLUT4-containing vesicles. (Affymetrix). Briefly, from three different sets of 3T3-Ll adipocytes, Recent work has identified potential receptors for the TPR mRNA was isolated using Oligotex mRNA kit (Qiagen). Double-stranded cDNA was synthesized from 5 µg mRNA, purified and biotinylated. The domains of kinesin light chains, including IlPl (Verhey cDNA was hybridized to the murine genome U74 arrays (A and B) for et al., 2001), Sunday Driver (SYD or IlP3) (Bowman et al., 16 h at 45 °C. The arrays were stained with streptavidin phycoerythrin 2000) and amyloid precursor protein (APP) (Kamal et al., solution and scanned in an HP GeneArray scanner. Average signal was 2001). Furthermore, the interaction between kinesin and calculated by subtracting signals from 11-20 overlapping 25mer probes cargo can be regulated with other proteins, such as heat by those from corresponding mismatch probes. shock chaperone 70 (Tsai et al., 2000). Also, recent work lmmunoprecipitation, mass spectrometry and western has implicated the protein kinase GSK3 as a regulator of blotting kinesin light chain interaction with membranes (Morfini Immunoprecipitation and mass spectrometry were performed as et al., 2002). These findings suggest a mechanism whereby described previously (Park et al., 2001) with 1 mg total protein and 4 µg antibodies. For western blotting, cell lysates were collected in SDS phosphorylation of kinesin light chain by GSK3 disrupts lysis buffer (20 mM HEPES, pH 7 .2, 1 % sodium dodecyl sulfate, 1 mM its interaction with receptors on membrane organelles. The sodium vanadate) supplemented with protease inhibitors and loaded onto role of phosphorylation in regulating kinesin function was 10% polyacrylarnide gels. also suggested by others (Lee and Hollenbeck, 1995; lmmunofluorescence microscopy De Vos et al., 2000; Morfini et al., 2001). This model, 3T3-Ll adipocytes were fixed with 4% formaldehyde in phosphate­ coupled with our data presented here, suggest there may be buffered saline (PBS), permeabilized and blocked with 0.5% Triton one or more receptors on GLUT4-containing vesicles that X-100 and 1 % fetal bovine serum in PBS for 20 min. Cells were bind the kinesin light chain TPR domain and are poten­ incubated with primary antibodies for 2 h and with rhodarnine- or FITC­ tially regulated by insulin signaling. Future experiments in conjugated secondary antibodies for 30 min. To analyze Myc-GLUT4- EGFP translocation in adipocytes, cells were immunostained as described our laboratory are directed to test this hypothesis. previously (Jiang et al., 2002). Briefly, the cell-surface Myc-GLUT4- GFP was visualized with anti-Myc antibody and rhodamine-labeled anti­ mouse secondary antibody without permeabilization. Where applicable, the adipocytes were then fixed, permeabilized and immunostained for Materials and methods HA-tagged proteins, using polyclonal anti-HA antibody and Alexa-350- conjugated secondary antibody. Images were taken with an Olympus IX- 70 microscope with CCD camera and then processed using Metamorph Materials software. The specific plasma membrane content of Myc-GLUT4-GFP Plasmids encoding HA-tagged rat KLCI-C and its truncated constructs, was measured as described previously (Jiang et al., 2002). For F-actin and HA-KLC-176 and HA-KLC-TPR6, were kindly provided by Dr mitochondrial staining, cells were immunostained with anti-HA antibody K.J.Verhey (Harvard Medical School). Myc-GLUT4-EGFP construct and FITC-conjugated secondary antibody. Rhodarnine-phalloidin was was prepared as described previously (Jiang et al., 2002). GLUT4-YFP added together with the secondary antibody. Mitotracker (Molecular was constructed by inserting Myc-GLUT4 into pBluescript containing a Probes) was added before initial fixation. linker with unique sites (Nhel and Agel), and then by inserting it into the same sites of pECFP-Cl vector (Clontech). For the tubulin--CFP Supplementary data construct, rat tubulin cDNA (DDBJ/EMBUGenBank accession No. Supplementary data are available at The EMBO Journal Online. NM_0ll6 55) was isolated and amplified from total RNA using standard RT-PCR. 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Journal

The EMBO Journal – Springer Journals

Published: May 15, 2003

Keywords: adipocytes; GLUT4; insulin; kinesin; microtubules

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