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ANALYSIS OF CARBON FILM PLANARITY BY REFLECTED LIGHT MICROSCOPY

ANALYSIS OF CARBON FILM PLANARITY BY REFLECTED LIGHT MICROSCOPY DES La tacrine (1,2,3,4-tbtrahydro-9-aminoacridine hydrochloride) est le premier mtdicament reconnu pour amkliorer I’Ctat des patients atteints de la form and Inhlbilion bv srlecific inhibitors the liver. activaled and maladie d’Alzheimer. Elle agit comme inhibitcur de I’ac&ylcholinesttrase proliferall~c hcpatic~s&late cells (HSCj are tt;e main source of type IV dans tes neurones cholinergiques centraux. Cependant, son administration collagc~~nsc gclatinnsc (MMP2). A membrane-type MMP (MT-MMP), with provoque une &%vation r&etsible des transaminases skriques, selon toute a Iranslnerrll~r;lnc domain, has been reported lo form a tri-molecular complex wilh MMPZ and TJMP2 leading to the activation of nroMMP2. No vraiscmblance suite g des l&ions hkpatiques, chez 30 I SO% des patients sucl; activation mechanism was descr%ed uptodate in the liver and [hc trait&s. Le mecanisme par lequel ce compose induit une hCpatotoxicitC n’est function of Ia~rnt MMP? secreted by activated HSC remains unclear. The pas encore tlucidC. La formation de mCtabolites toxiques par le cytochrome goal of Ihi< work was II? study the cooperation between cultured stellalc P4SO (CYP) IA ainsi qu’une inhibition de la synthtse prottique par cells anti hroatocylcs 111 exlracellular malrix degradation. After a few days agrkgation des ribosomes ont CtC propos8es. Nous avons BtudiC les effets de in pi in1.11~ ~.ullurcs. I-ISC become “activated” and express extmcellula~ la tacrine sur des htpatocytes de rat et d’homme en culture primaire ainsi maIris co~nponcn~s AI high levels. Expression of MMPZ, TJMP2 and MT- que sur des cellules hCpatiques et non hCpatiques n’exprimant pas le MM1 IIIRNA \v:I< s~r~d~rtl both hy Norihcrn blot and in situ hvbridizarion. CYPIA. Quel que soit le type cellulaire consid&@ la tacrine est cytotoxique Grla~~nr~ly~~ acllvillc> WI-C anal&d by zymography. Rcsul& : Northern hlot ;LII;II~SI\and in \rttr hybridizaf~on showed thaf MMPZ, TJMJ’Z and MT- g des concentrations bgales ou supkrieures g 0,25mM aprks 20h de hIMI IIIK~“:\ \VCIC cspressed by ac~ivatcd hepalic slellale cells and no1 traitement, et des altirations- spbcifiques des ribosomes (formation hcpal~~~~~lc\.in ho111putt and cocutlules. Bo\h Ialenl aiid xlivatcd forms 01 d’agrkgats) sont observCes aprBs une exposition coulte (45min g 2h) en MhlJ’2 ;\c~c pcscnl In condilioncd media from coculiurcs system whci-c;ls prCsence de 1mM ou plus. Des traitements quofidiens pendant 2 jours pure CIII~IIIC~of t ISC sccrclcd only hltent MMP2. No gelatinolytic activio entrainent une inhibition de la neosynthbse protCique et une diminution du wa\ tlclccl~,~l 111 contl~tionnett nicdia FIorn hepatocyre cullurcs. Incubation oi taux de glycogtne B des concentrations de 0,05 B 0,JmM en absence plasma I~ICIII~~I-.IIIC ictlett fraction of hcpaiocy& with conditioned media d’altkrations de la morphologie des h$atocytes. Les rCsultats sugghent que cn~ from I IX‘ pcncralcd two lx~nds of aclivnted MMP2 (Mr = 62 kD and 59 la toxicit& de la tacrine chez I’homme n’est corr&e ni g son mCtabolisme LI)) l‘lr~\ pli~\i~u mcn~l~li~nc tlct~cntlanl aclivatioll mcchimism was royalty oxydatif ni & I’agrtgation des ribosomes (qui nc survient qu’g des I~~II~I(cII bv t:l)TA bllt not bv serinc orolcases inhibitors (PMSI;. NEM). concentrations BlevCes). Elle est plus vraisembiablement associ6e g une ‘I‘f ypsin Ir~;~lr~~cr~l ot heating plasu;n nlcmbrane prcpnkions hefurc incrlhatm~~ ~~ICVCIIIC~ lhe x~~vat~on of proMMP2. Conclusion : Coculmre of perturbation de certaines voies mCtaboliques nkcessitant un apport en IJSC wIltI hcpa~ocy~cs iriduced both a deposition of matrix components ant] Bnergie (Etude supportCe par I’INSERM et la CEE). ~C~I\~IIW 01. MMJ’2 Coordina~cd expression of MT-MMP. MMP2 an<1 TIMl’2 by hcpalic slctlatc cells was nol sufficient to the release of an acl~va~cd hIMI’?. l’hrls. hepalocyles might induce rhe activation of proM hl I? (_I on, cullurcd FJSC via a plasma membrane dcpcndanr mc~han~rm \vhIch could result from either conformalional changes of Ihc putalive II-IIIII~ICCIIIIII Icomplex (MT-MMP/MMP2/TlMP2) or a molt comptcs ~IIOCCSS involving cell surface proteases. is rcgul;&l ;U rhrcc levels : gem expression, iktivalion (TJMP). In of ihe secreted latenr Nathalic ThCret, Orlando Musso. Anme L’tlelgounl’ch, Bruno CICmcnt. Groups “DPloxication et RCoaration Tissulaire” INSERM. CI-IRU Pontcila~th. 3.5033 RENNES, $RA~VCE Matrix Metalloproleinases (MMP) are a family of enzymes involved in the dcrradation of extracellular matrix comnonents. The activilv of MMP RISSEL Mary, LAG&XC-GOSSMANN Dominique, GUILLOUZO Andrt Scietms drt Pr($ Groupe LMoxication et R+ara/ion Tissulawe, I~aculfC des I’harmaceutiqrres et Biologiques, Uuh~ersitP de Rermes I, A veme L&OH Bernard, 35043 Retmes cedex, Frame ULTRARAPID FREEZING (OUICK-FREEZE PRESSURE) APPLIED TO RE;CONSTITUTED STUDY IN TEM AFTER FREEZE-ETCHING. 01~ HIGHLIPID BODIES ANALYSIS OF CARBON LIGHT MICROSCOPY FILM PLANARITY BY REFLECTED LECHAIRElJean-Pierre, MARULL2 Sylvie and GAILLiFran$oise I 11 CNRS CIPR 9042/CIME-Jussierr Urziversite’ P. et M. Cwie, 7 Qmi St Bemnrcl 75252 Paris Cedes 05 (France). (2) Centrr de Recherclk Yves Rocller 7-9, Av. F. V. Ra.yni/ 94117 Arcueil Cedex (Frmce) Schmutz Marc and Brisson Alain# IGBMC, UlM-JNSERM, CNRS, UniversitC Louis Pasteur, ColJ?ge de France BP 163 F67404 Illkirch Cedex #present address: University of Groningen-Department of Chemistry Biophysical Chemistry, Nijenborgh 4, NL-9747 AG Groningen. Defects of planarity of two-dimensional crystals constitute a major limitation in high resolution structural studies of biological macromolecules by electron crystallography. We found that reflected light microscopy constitutes a simple and appropriate technique for revealing the state of planarity of carbon films which are-commonly used for suooortine soecimens in electron microscoov (Schmulz M., Lang J., Graff S.:‘and &i&on A. (1994). J. Struct. Bidt:, 112. 252-258). Carbon films evaoorated on mica and floated on a water surface presented straight striations’aligned in parallel se& and extending over large distances. Wrinkles were frequently observed on one side of these striations. After transfer to electron microscope grids, carbon films exhibited breaks with a oattern similar to that of the striations. In addition. a second type of wrinkles, not related to the striations, was observed covering a major surface of everv grid. The wrinkles could be detected bv transmission > electron microscopy at extremely high values of defocusing, by scanning electron microscopy using highly tilted specimens, and it was emphasised by Nomarski differential interference contrast using reflected light microscopy. In our attempts to limit the extent of wrinkling, we found that the side of electron microscope grids on which carbon fi‘ilms were transferred had a preponderant influence on the wrinkling process. The dull side gave When Plunge-freezing was used, we observed irregular vesicles, a consistently a better result than the shiny side. This effect was correlated with damaged aspect Ef the surrounding matrix by ice cristals, and few scanning electron microscopy observations. Nitrocellulose films floated on a information concerning the orotein distribution. QF and I-IP freezing water surface as well as carbon coated nitrocellulose films deuosited on techniques gave complementarjr structural informations concerning the lipid electron microscope grids were frequently obtained devoid of breaks and body matrix, its peripherical protein arrangement and the surrounding matrix wrinkles. Dissolution of nitrocellulose was accompanied bv the aooarition of The depth of good freezing being dependent of the distance to the cooling small wrinkles. We also investigated the wrinkling of carbon-coaie’d grids by surface, for HP freezing the standard gold platelets with an important wall freezing in liquid nitrogen, in relationship with the use of cryo-techniques in thickness and deep cavities were not suitable for our application. We used electron crystallography of biological macromolecules. Freezing flat, plain carbon films resulted in a reversible cryo-induced wrinkling, in agreement specially fabricated thinner wall copper platelets with flatter depressions. These results permit to discuss the model proposed for the oleosomes with original observations from Boov and Pawlev (Boov F. P. and Pawtev J. B. (1993). Ultramicroscopy, 48, 275-280). No dr;o in&ed wrinkling &as (Tzen J.T.C., Huang A.H.C. (1992). J. Cell Biol., 117.327-335) observed after nitrocellulose dissolution, although these films were significantly less fragile than plain carbon films (Schmutz M; and Brisson A. Ultramicroscopy ir~press). Reconstituted lipid bodies, extracted from almonds, form spherical vesicles. These vesicle,; have an oil matrix surrounded by an half unit membrane composed of phospholipids and proteins (termed ileosins). Their structural studv in TEM is poorly known, and the few reports concern o111y the native lipi bodies (ole&om&) after chemical fixati&. It is known that chemical fixation and dehydratation procedures may introduce extensive structural artifacts, particularly in 1ge case of lipid containing material (Aneerbeck L.P.. Gulik-Krzvwiki T. (1986). Methods in Enzymology, 128, 4577472) as reconstituted lipid bodies: Related to the studied-structLi;es and particularly lo the protein distribution, cryofixations seemed to be more convenient. It is known that the Quick-freezing technique allows a good fixation only in the few micrometer depth (5-t 0 em) from the sample surface (Favard P., Lechaire J.P , Maillard M., Favard N., Djabourov M., Leblond J. (1989), Biol. Cell.. 67, 201-207). Whereas the High-pressure freezing permits good structural preservation up to 200 /ini depth (I-Johenberg Id., Tobler M.. Miilter M. (; 994) JCEM 13 Paris, 955-986). According lo these considerations we compared three techniques: Plunge-freezing (Freon). Quick-freezing (QF) anc! High-pressure (J{P) freezing. > Y http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Biology of the Cell Wiley

ANALYSIS OF CARBON FILM PLANARITY BY REFLECTED LIGHT MICROSCOPY

Marc, Schmutz; Alain, Brisson
Biology of the Cell , Volume 86 (2‐3) – Jan 1, 1996
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Publisher
Wiley
Copyright
1996 Société Française des Microscopies and Société Biologie Cellulaire de France
ISSN
0248-4900
eISSN
1768-322X
DOI
10.1016/0248-4900(96)84798-8
Publisher site
See Article on Publisher Site

Abstract

DES La tacrine (1,2,3,4-tbtrahydro-9-aminoacridine hydrochloride) est le premier mtdicament reconnu pour amkliorer I’Ctat des patients atteints de la form and Inhlbilion bv srlecific inhibitors the liver. activaled and maladie d’Alzheimer. Elle agit comme inhibitcur de I’ac&ylcholinesttrase proliferall~c hcpatic~s&late cells (HSCj are tt;e main source of type IV dans tes neurones cholinergiques centraux. Cependant, son administration collagc~~nsc gclatinnsc (MMP2). A membrane-type MMP (MT-MMP), with provoque une &%vation r&etsible des transaminases skriques, selon toute a Iranslnerrll~r;lnc domain, has been reported lo form a tri-molecular complex wilh MMPZ and TJMP2 leading to the activation of nroMMP2. No vraiscmblance suite g des l&ions hkpatiques, chez 30 I SO% des patients sucl; activation mechanism was descr%ed uptodate in the liver and [hc trait&s. Le mecanisme par lequel ce compose induit une hCpatotoxicitC n’est function of Ia~rnt MMP? secreted by activated HSC remains unclear. The pas encore tlucidC. La formation de mCtabolites toxiques par le cytochrome goal of Ihi< work was II? study the cooperation between cultured stellalc P4SO (CYP) IA ainsi qu’une inhibition de la synthtse prottique par cells anti hroatocylcs 111 exlracellular malrix degradation. After a few days agrkgation des ribosomes ont CtC propos8es. Nous avons BtudiC les effets de in pi in1.11~ ~.ullurcs. I-ISC become “activated” and express extmcellula~ la tacrine sur des htpatocytes de rat et d’homme en culture primaire ainsi maIris co~nponcn~s AI high levels. Expression of MMPZ, TJMP2 and MT- que sur des cellules hCpatiques et non hCpatiques n’exprimant pas le MM1 IIIRNA \v:I< s~r~d~rtl both hy Norihcrn blot and in situ hvbridizarion. CYPIA. Quel que soit le type cellulaire consid&@ la tacrine est cytotoxique Grla~~nr~ly~~ acllvillc> WI-C anal&d by zymography. Rcsul& : Northern hlot ;LII;II~SI\and in \rttr hybridizaf~on showed thaf MMPZ, TJMJ’Z and MT- g des concentrations bgales ou supkrieures g 0,25mM aprks 20h de hIMI IIIK~“:\ \VCIC cspressed by ac~ivatcd hepalic slellale cells and no1 traitement, et des altirations- spbcifiques des ribosomes (formation hcpal~~~~~lc\.in ho111putt and cocutlules. Bo\h Ialenl aiid xlivatcd forms 01 d’agrkgats) sont observCes aprBs une exposition coulte (45min g 2h) en MhlJ’2 ;\c~c pcscnl In condilioncd media from coculiurcs system whci-c;ls prCsence de 1mM ou plus. Des traitements quofidiens pendant 2 jours pure CIII~IIIC~of t ISC sccrclcd only hltent MMP2. No gelatinolytic activio entrainent une inhibition de la neosynthbse protCique et une diminution du wa\ tlclccl~,~l 111 contl~tionnett nicdia FIorn hepatocyre cullurcs. Incubation oi taux de glycogtne B des concentrations de 0,05 B 0,JmM en absence plasma I~ICIII~~I-.IIIC ictlett fraction of hcpaiocy& with conditioned media d’altkrations de la morphologie des h$atocytes. Les rCsultats sugghent que cn~ from I IX‘ pcncralcd two lx~nds of aclivnted MMP2 (Mr = 62 kD and 59 la toxicit& de la tacrine chez I’homme n’est corr&e ni g son mCtabolisme LI)) l‘lr~\ pli~\i~u mcn~l~li~nc tlct~cntlanl aclivatioll mcchimism was royalty oxydatif ni & I’agrtgation des ribosomes (qui nc survient qu’g des I~~II~I(cII bv t:l)TA bllt not bv serinc orolcases inhibitors (PMSI;. NEM). concentrations BlevCes). Elle est plus vraisembiablement associ6e g une ‘I‘f ypsin Ir~;~lr~~cr~l ot heating plasu;n nlcmbrane prcpnkions hefurc incrlhatm~~ ~~ICVCIIIC~ lhe x~~vat~on of proMMP2. Conclusion : Coculmre of perturbation de certaines voies mCtaboliques nkcessitant un apport en IJSC wIltI hcpa~ocy~cs iriduced both a deposition of matrix components ant] Bnergie (Etude supportCe par I’INSERM et la CEE). ~C~I\~IIW 01. MMJ’2 Coordina~cd expression of MT-MMP. MMP2 an<1 TIMl’2 by hcpalic slctlatc cells was nol sufficient to the release of an acl~va~cd hIMI’?. l’hrls. hepalocyles might induce rhe activation of proM hl I? (_I on, cullurcd FJSC via a plasma membrane dcpcndanr mc~han~rm \vhIch could result from either conformalional changes of Ihc putalive II-IIIII~ICCIIIIII Icomplex (MT-MMP/MMP2/TlMP2) or a molt comptcs ~IIOCCSS involving cell surface proteases. is rcgul;&l ;U rhrcc levels : gem expression, iktivalion (TJMP). In of ihe secreted latenr Nathalic ThCret, Orlando Musso. Anme L’tlelgounl’ch, Bruno CICmcnt. Groups “DPloxication et RCoaration Tissulaire” INSERM. CI-IRU Pontcila~th. 3.5033 RENNES, $RA~VCE Matrix Metalloproleinases (MMP) are a family of enzymes involved in the dcrradation of extracellular matrix comnonents. The activilv of MMP RISSEL Mary, LAG&XC-GOSSMANN Dominique, GUILLOUZO Andrt Scietms drt Pr($ Groupe LMoxication et R+ara/ion Tissulawe, I~aculfC des I’harmaceutiqrres et Biologiques, Uuh~ersitP de Rermes I, A veme L&OH Bernard, 35043 Retmes cedex, Frame ULTRARAPID FREEZING (OUICK-FREEZE PRESSURE) APPLIED TO RE;CONSTITUTED STUDY IN TEM AFTER FREEZE-ETCHING. 01~ HIGHLIPID BODIES ANALYSIS OF CARBON LIGHT MICROSCOPY FILM PLANARITY BY REFLECTED LECHAIRElJean-Pierre, MARULL2 Sylvie and GAILLiFran$oise I 11 CNRS CIPR 9042/CIME-Jussierr Urziversite’ P. et M. Cwie, 7 Qmi St Bemnrcl 75252 Paris Cedes 05 (France). (2) Centrr de Recherclk Yves Rocller 7-9, Av. F. V. Ra.yni/ 94117 Arcueil Cedex (Frmce) Schmutz Marc and Brisson Alain# IGBMC, UlM-JNSERM, CNRS, UniversitC Louis Pasteur, ColJ?ge de France BP 163 F67404 Illkirch Cedex #present address: University of Groningen-Department of Chemistry Biophysical Chemistry, Nijenborgh 4, NL-9747 AG Groningen. Defects of planarity of two-dimensional crystals constitute a major limitation in high resolution structural studies of biological macromolecules by electron crystallography. We found that reflected light microscopy constitutes a simple and appropriate technique for revealing the state of planarity of carbon films which are-commonly used for suooortine soecimens in electron microscoov (Schmulz M., Lang J., Graff S.:‘and &i&on A. (1994). J. Struct. Bidt:, 112. 252-258). Carbon films evaoorated on mica and floated on a water surface presented straight striations’aligned in parallel se& and extending over large distances. Wrinkles were frequently observed on one side of these striations. After transfer to electron microscope grids, carbon films exhibited breaks with a oattern similar to that of the striations. In addition. a second type of wrinkles, not related to the striations, was observed covering a major surface of everv grid. The wrinkles could be detected bv transmission > electron microscopy at extremely high values of defocusing, by scanning electron microscopy using highly tilted specimens, and it was emphasised by Nomarski differential interference contrast using reflected light microscopy. In our attempts to limit the extent of wrinkling, we found that the side of electron microscope grids on which carbon fi‘ilms were transferred had a preponderant influence on the wrinkling process. The dull side gave When Plunge-freezing was used, we observed irregular vesicles, a consistently a better result than the shiny side. This effect was correlated with damaged aspect Ef the surrounding matrix by ice cristals, and few scanning electron microscopy observations. Nitrocellulose films floated on a information concerning the orotein distribution. QF and I-IP freezing water surface as well as carbon coated nitrocellulose films deuosited on techniques gave complementarjr structural informations concerning the lipid electron microscope grids were frequently obtained devoid of breaks and body matrix, its peripherical protein arrangement and the surrounding matrix wrinkles. Dissolution of nitrocellulose was accompanied bv the aooarition of The depth of good freezing being dependent of the distance to the cooling small wrinkles. We also investigated the wrinkling of carbon-coaie’d grids by surface, for HP freezing the standard gold platelets with an important wall freezing in liquid nitrogen, in relationship with the use of cryo-techniques in thickness and deep cavities were not suitable for our application. We used electron crystallography of biological macromolecules. Freezing flat, plain carbon films resulted in a reversible cryo-induced wrinkling, in agreement specially fabricated thinner wall copper platelets with flatter depressions. These results permit to discuss the model proposed for the oleosomes with original observations from Boov and Pawlev (Boov F. P. and Pawtev J. B. (1993). Ultramicroscopy, 48, 275-280). No dr;o in&ed wrinkling &as (Tzen J.T.C., Huang A.H.C. (1992). J. Cell Biol., 117.327-335) observed after nitrocellulose dissolution, although these films were significantly less fragile than plain carbon films (Schmutz M; and Brisson A. Ultramicroscopy ir~press). Reconstituted lipid bodies, extracted from almonds, form spherical vesicles. These vesicle,; have an oil matrix surrounded by an half unit membrane composed of phospholipids and proteins (termed ileosins). Their structural studv in TEM is poorly known, and the few reports concern o111y the native lipi bodies (ole&om&) after chemical fixati&. It is known that chemical fixation and dehydratation procedures may introduce extensive structural artifacts, particularly in 1ge case of lipid containing material (Aneerbeck L.P.. Gulik-Krzvwiki T. (1986). Methods in Enzymology, 128, 4577472) as reconstituted lipid bodies: Related to the studied-structLi;es and particularly lo the protein distribution, cryofixations seemed to be more convenient. It is known that the Quick-freezing technique allows a good fixation only in the few micrometer depth (5-t 0 em) from the sample surface (Favard P., Lechaire J.P , Maillard M., Favard N., Djabourov M., Leblond J. (1989), Biol. Cell.. 67, 201-207). Whereas the High-pressure freezing permits good structural preservation up to 200 /ini depth (I-Johenberg Id., Tobler M.. Miilter M. (; 994) JCEM 13 Paris, 955-986). According lo these considerations we compared three techniques: Plunge-freezing (Freon). Quick-freezing (QF) anc! High-pressure (J{P) freezing. > Y

Journal

Biology of the Cell – Wiley

Published: Jan 1, 1996

There are no references for this article.