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Edward D. Korn - One of the best experts on this subject based on the ideXlab platform.
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dynamIc localIzatIon of MyosIn I to endocytIc structures In acanthamoeba
Cytoskeleton, 2003Co-Authors: Michael E Ostap, Ivan C. Baines, Edward D. Korn, Pamela Maupin, Steven K. Doberstein, Thomas D. PollardAbstract:We used fluorescence mIcroscopy of lIve Acanthamoeba to follow the tIme course of the concentratIon of MyosIn-I next to the plasma membrane at sItes of macropInocytosIs and phagocytosIs. We marked MyosIn-I wIth a fluorescently labeled monoclonal antIbody (Cy3-M1.7) Introduced Into the cytoplasm by syrInge loadIng. M1.7 bInds MyosIn-IA and -IC wIthout affectIng theIr actIvItIes, but does not bInd MyosIn-IB. Cy3-M1.7 concentrates at two dIfferent macropInocytIc structures: large cIrcular membrane ruffles that fuse to create macropInosomes, and smaller endocytIc structures that occur at the end of stalk-lIke pseudopodIa. These dynamIc structures enclose macropInosomes every 30-60 s. Cy3-M1.7 accumulates rapIdly as these endocytIc structures form and dIssIpate rapIdly after they InternalIze. Double labelIng fIxed cells wIth Cy3-M1.7 and polyclonal antIbodIes specIfIc for MyosIn-IA, -IB, or -IC revealed that all three MyosIn-I Isoforms assocIate wIth macropInocytIc structures, but IndIvIdual structures vary In theIr MyosIn-I Isoform composItIon. MyosIn-I and actIn also concentrate transIently at sItes where amoebae Ingest yeast or the pseudopodIa of neIghborIng cells (heterophagy) by the process of phagocytosIs. WIthIn 3 mIn of yeast attachment to the amoeba, MyosIn-I concentrates around the phagocytIc cup, yeast are InternalIzed, and MyosIn-I de-localIzes. DespIte known dIfferences In the regulatIon of macropInocytosIs and phagocytosIs, the morphology, proteIn composItIon, and dynamIcs of phagocytosIs and macropInocytosIs are sImIlar, IndIcatIng that they share common structural propertIes and contractIle mechanIsms.
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DynamIc localIzatIon of MyosIn‐I to endocytIc structures In Acanthamoeba
Cell motility and the cytoskeleton, 2002Co-Authors: E. Michael Ostap, Ivan C. Baines, Edward D. Korn, Pamela Maupin, Steven K. Doberstein, Thomas D. PollardAbstract:We used fluorescence mIcroscopy of lIve Acanthamoeba to follow the tIme course of the concentratIon of MyosIn-I next to the plasma membrane at sItes of macropInocytosIs and phagocytosIs. We marked MyosIn-I wIth a fluorescently labeled monoclonal antIbody (Cy3-M1.7) Introduced Into the cytoplasm by syrInge loadIng. M1.7 bInds MyosIn-IA and -IC wIthout affectIng theIr actIvItIes, but does not bInd MyosIn-IB. Cy3-M1.7 concentrates at two dIfferent macropInocytIc structures: large cIrcular membrane ruffles that fuse to create macropInosomes, and smaller endocytIc structures that occur at the end of stalk-lIke pseudopodIa. These dynamIc structures enclose macropInosomes every 30-60 s. Cy3-M1.7 accumulates rapIdly as these endocytIc structures form and dIssIpate rapIdly after they InternalIze. Double labelIng fIxed cells wIth Cy3-M1.7 and polyclonal antIbodIes specIfIc for MyosIn-IA, -IB, or -IC revealed that all three MyosIn-I Isoforms assocIate wIth macropInocytIc structures, but IndIvIdual structures vary In theIr MyosIn-I Isoform composItIon. MyosIn-I and actIn also concentrate transIently at sItes where amoebae Ingest yeast or the pseudopodIa of neIghborIng cells (heterophagy) by the process of phagocytosIs. WIthIn 3 mIn of yeast attachment to the amoeba, MyosIn-I concentrates around the phagocytIc cup, yeast are InternalIzed, and MyosIn-I de-localIzes. DespIte known dIfferences In the regulatIon of macropInocytosIs and phagocytosIs, the morphology, proteIn composItIon, and dynamIcs of phagocytosIs and macropInocytosIs are sImIlar, IndIcatIng that they share common structural propertIes and contractIle mechanIsms.
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p21-actIvated kInase has substrate specIfIcIty sImIlar to Acanthamoeba MyosIn I heavy chaIn kInase and actIvates Acanthamoeba MyosIn I
Proceedings of the National Academy of Sciences of the United States of America, 1997Co-Authors: Hanna Brzeska, Ulla G. Knaus, Zhen-yuan Wang, Gary M. Bokoch, Edward D. KornAbstract:Acanthamoeba class I MyosIns are unconventIonal, sIngle-headed MyosIns that express actIn-actIvated Mg2+-ATPase and In vItro motIlIty actIvItIes only when a sIngle serIne or threonIne In the heavy chaIn Is phosphorylated by MyosIn I heavy chaIn kInase (MIHCK). Some other, but not most, class I MyosIns have the same consensus phosphorylatIon sIte sequence, and the two known class VI MyosIns have a phosphorylatable resIdue In the homologous posItIon, where most MyosIns have an aspartate or glutamate resIdue. Recently, we found that the catalytIc domaIn of Acanthamoeba MIHCK has extensIve sequence sImIlarIty to the p21-actIvated kInase (PAK)/STE20 famIly of kInases from mammals and yeast, whIch are actIvated by small GTP-bIndIng proteIns. The physIologIcal substrates of the PAK/STE20 kInases are not well characterIzed. In thIs paper we show that PAK1 has sImIlar substrate specIfIcIty as MIHCK when assayed agaInst synthetIc substrates and that PAK1 phosphorylates the heavy chaIn (1 mol of PI per mol) and actIvates Acanthamoeba MyosIn I as MIHCK does. These results, together wIth the known Involvement of Acanthamoeba MyosIn I, yeast MyosIn I, STE20, PAK, and small GTP-bIndIng proteIns In membrane- and cytoskeleton-assocIated morphogenetIc transformatIons and actIvItIes, suggest that MyosIns may be physIologIcal substrates for the PAK/STE20 famIly and thus medIators of these events.
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PropertIes of Acanthamoeba MyosIn I heavy chaIn kInase bound to phospholIpId vesIcles.
The Journal of biological chemistry, 1995Co-Authors: Zhen-yuan Wang, Hanna Brzeska, Ivan C. Baines, Edward D. KornAbstract:The actIn-actIvated Mg(2+)-ATPase and In vItro motIlIty actIvItIes of the three Acanthamoeba MyosIn I Isozymes depend upon phosphorylatIon of theIr sIngle heavy chaIns by MyosIn I heavy chaIn kInase. PrevIously, the kInase had been shown to be actIvated by autophosphorylatIon, whIch Is enhanced by acIdIc phospholIpIds, or sImply by bIndIng to purIfIed plasma membranes In the absence of sIgnIfIcant autophosphorylatIon. In thIs paper, we show that the rate of phosphorylatIon of MyosIn I by unphosphorylated kInase Is approxImately 20-fold faster when both the MyosIn I and the kInase are bound to acIdIc phospholIpId vesIcles than when both are soluble. ThIs actIvatIon Is not due to an Increase In the local concentratIons of vesIcle-bound kInase and MyosIn I. Thus, acIdIc phospholIpIds, lIke membranes, can actIvate MyosIn I heavy chaIn kInase In the absence of sIgnIfIcant autophosphorylatIon, I.e. membrane proteIns are not requIred. KInetIc studIes show that both bIndIng of kInase to phospholIpId vesIcles and autophosphorylatIon of kInase In the absence of phospholIpId Increase the Vmax relatIve to soluble, unphosphorylated kInase wIth eIther an Increase In the apparent Km (when MyosIn I Is the substrate) or no sIgnIfIcant change In Km (when a synthetIc peptIde Is the substrate). KInetIc data showed that autophosphorylatIon of phospholIpId-bound kInase Is both Intermolecular and IntervesIcular, and that phosphorylatIon of phospholIpId-bound MyosIn I by phospholIpId-bound kInase Is also IntervesIcular even when the kInase and MyosIn are bound to the same vesIcles. The relevance of these results to the actIvatIon of MyosIn I heavy chaIn kInase and phosphorylatIon of MyosIn I Isozymes In sItu are dIscussed.
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QuantIfIcatIon and localIzatIon of phosphorylated MyosIn I Isoforms In Acanthamoeba castellanII.
The Journal of cell biology, 1995Co-Authors: Ivan C. Baines, A Corigliano-murphy, Edward D. KornAbstract:The actIn-actIvated Mg(2+)-ATPase actIvItIes of the three MyosIn I Isoforms In Acanthamoeba castellanII are sIgnIfIcantly expressed only after phosphorylatIon of a sIngle sIte In the MyosIn I heavy chaIn. SynthetIc phosphorylated and unphosphorylated peptIdes correspondIng to the phosphorylatIon sIte sequences, whIch dIffer for the three MyosIn I Isoforms, were used to raIse Isoform-specIfIc antIbodIes that recognIzed only the phosphorylated MyosIn I or the total MyosIn I Isoform (phosphorylated and unphosphorylated), respectIvely. WIth these antIsera, the amounts of total and phosphorylated Isoform were quantIfIed, the phosphoMyosIn I Isoforms localIzed, and the compartmental dIstrIbutIon of the phosphoMyosIn Isoforms determIned. MyosIn IA, whIch was almost entIrely In the actIn-rIch cortex, was 70-100% phosphorylated and partIcularly enrIched under phagocytIc cups. MyosIns IB and IC were predomInantly assocIated wIth plasma membranes and large vacuole membranes, where they were only 10-20% phosphorylated, whereas cytoplasmIc MyosIns IB and IC, lIke cytoplasmIc MyosIn IA, were mostly phosphorylated (60-100%). Moreover, phosphoMyosIn IB was concentrated In actIvely motIle regIons of the plasma membrane. More than 20-fold more phosphoMyosIn IC and 10-fold more F-actIn were assocIated wIth the membranes of contractIng contractIle vacuoles (CV) than of fIllIng CVs. As the total amount of CV-assocIated MyosIn IC remaIned constant, It must be phosphorylated at the start of CV contractIon. These data extend prevIous proposals for the specIfIc functIons of MyosIn I Isozymes In Acanthamoeba (BaInes, I.C., H. Brzeska, and E.D. Korn. 1992. J. Cell BIol. 119: 1193-1203): phosphoMyosIn IA In phagocytosIs, phosphoMyosIn IB In phagocytosIs and pInocytosIs, and phosphoMyosIn IC In contractIon of the CV.
Ekkehard Leberer - One of the best experts on this subject based on the ideXlab platform.
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MyosIn I Is RequIred for Hypha FormatIon In CandIda albIcans
Eukaryotic cell, 2002Co-Authors: Ursula Oberholzer, Anne Marcil, Ekkehard Leberer, David Y. Thomas, Malcolm WhitewayAbstract:PolarIzed growth Is a regulated cellular expansIon whIch underlIes many processes, such as phagocytosIs In mammalIan cells, morphogenesIs of root haIr and other specIalIzed cell types In plants, cell locomotIon In Acanthamoeba and DIctyostelIum, and hypha formatIon In fungI (19, 24, 37, 49). Saccharomyces cerevIsIae cells can elongate Into pseudohyphae In response to specIfIc envIronmental cues, and polarIzatIon of the actIn cytoskeleton Is essentIal for thIs dIfferentIatIon (11). SImIlarly, hyphal morphogenesIs In other fungI, such as SaprolegnIa ferax, Neurospora crassa, and AspergIllus nIdulans, requIres fIlamentous actIn, whIle mIcrotubules play a secondary role (20, 51, 52). The pathogenIc yeast CandIda albIcans can undergo a dramatIc change In morphogenesIs when round yeast cells form hIghly elongated fIlaments called hyphae, but lIttle Is known about the role of the actIn cytoskeleton durIng hypha formatIon In thIs organIsm (25). Treatment of germInatIng cells wIth cytochalasIn A prevents further hyphal growth, suggestIng that fIlamentous actIn Is crItIcal to hyphal growth (2). Nocodazole, In contrast, does not prevent apIcal cell elongatIon, suggestIng that mIcrotubules are not crItIcal for polarIzed growth In C. albIcans (61). Two forms of actIn appear to be Important durIng polarIzed growth. FIrst, actIn cables serve as tracks for the vesIcular transport of molecular components of the plasma membrane and cell wall toward the sIte of growth, I.e., the bud tIp (23, 44, 45) and presumably the hyphal tIp of C. albIcans. These run along the longItudInal axIs of yeast and hyphal cells In S. cerevIsIae and C. albIcans (4, 44). Second, cortIcal actIn patches correlate wIth sItes of targeted secretIon and endocytosIs, crItIcal durIng cell wall bIogenesIs (44). These localIze to the tIps of emergIng buds as well to growIng hyphal tIps In C. albIcans (4). ProteIns that modulate the structure of the actIn cytoskeleton are key factors In determInIng cell polarIty (44). MyosIn I, one of these factors, Is a sIngle-headed molecular motor that functIons In actIn-based processes such as polarIzed growth, cell motIlIty, phagocytosIs, endocytosIs and exocytosIs, and contractIle vacuolar actIvIty In several organIsms (12, 13, 32, 37, 40, 46, 50, 58). In S. cerevIsIae, MyosIn I was shown to promote actIn polymerIzatIon at cortIcal patches, whIch correlate wIth sItes of growth (1, 14, 29). ThIs MyosIn I regulatIon of actIn polymerIzatIon was shown to be achIeved by Its InteractIon wIth and actIvatIon of the Arp2/3 complex, whIch nucleates the assembly of actIn fIlaments (14, 29, 33, 34). SImIlarly, In SchIzosaccharomyces pombe, MyosIn I Is requIred for a polarIzed actIn cytoskeleton and was shown to bInd to the Arp2/Arp3 complex and actIvate Its actIn nucleatIon actIvIty (32, 53). Jung et al. (22) also found that DyctIostelIum MyosIn I Interacts wIth the Arp2-Arp3 complex vIa the CARMIL proteIn and may localIze actIn polymerIzatIon to sItes of cellular growth. In accordance wIth Its proposed role In polarIzed growth, MyosIn I colocalIzes wIth cortIcal actIn patches at the tIps of buds In S. cerevIsIae and of growIng cells In S. pombe (3, 32, 53) and localIzes as well to the tIps of hyphae In AspergIllus (36). The actIn-dependent ATPase actIvIty of Acanthamoeba MyosIn I and DIctyostelIum MyosIn I Is actIvated by the phosphorylatIon of a unIque sIte, called the “TEDS-rule” sIte, by members of the p21-actIvated kInase (PAK) kInases (7, 9, 56). The correspondIng phosphorylatIon sIte of S. cerevIsIae MyosIn I Is essentIal for Its functIon In vIvo and for Its abIlIty to polymerIze actIn In vItro (29, 57). It Is also a target of the Ste20p and Cla4p kInases In vItro (57). These latter proteIns are members of the PAK famIly of proteIn kInases and functIon to regulate cell morphology (6, 27, 47). Homologues of these kInases are Involved In hypha formatIon In C. albIcans (8, 26, 28). In thIs study, we took a genetIc approach to defIne the role of MyosIn I In C. albIcans. We found that MyosIn I Is requIred for hypha but not pseudohypha formatIon and that the PAK phosphorylatIon sIte (serIne 366) Is crItIcal for MyosIn I functIon durIng buddIng yeast and hyphal growth.
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A Role for MyosIn-I In ActIn Assembly through InteractIons wIth Vrp1p, Bee1p, and the Arp2/3 Complex
The Journal of cell biology, 2000Co-Authors: Marie Evangelista, Malcolm Whiteway, Ekkehard Leberer, David Y. Thomas, Bert Klebl, Amy H.y. Tong, Bradley A. Webb, Thomas Leeuw, Charles BooneAbstract:Type I MyosIns are hIghly conserved actIn-based molecular motors that localIze to the actIn-rIch cortex and partIcIpate In motIlIty functIons such as endocytosIs, polarIzed morphogenesIs, and cell mIgratIon. The COOH-termInal taIl of yeast MyosIn-I proteIns, Myo3p and Myo5p, contaIns an Src homology domaIn 3 (SH3) followed by an acIdIc domaIn. The MyosIn-I SH3 domaIn Interacted wIth both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteIns that lInk actIn assembly and sIgnalIng molecules. The MyosIn-I acIdIc domaIn Interacted wIth Arp2/3 complex subunIts, Arc40p and Arc19p, and showed both sequence sImIlarIty and genetIc redundancy wIth the COOH-termInal acIdIc domaIn of Bee1p (Las17p), whIch controls Arp2/3-medIated actIn nucleatIon. These fIndIngs suggest that MyosIn-I proteIns may partIcIpate In a dIverse set of motIlIty functIons through a role In actIn assembly.
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a role for MyosIn I In actIn assembly through InteractIons wIth vrp1p bee1p and the arp2 3 complex
Journal of Cell Biology, 2000Co-Authors: Marie Evangelista, Malcolm Whiteway, Ekkehard Leberer, David Y. Thomas, Bert Klebl, Amy H.y. Tong, Bradley A. Webb, Thomas Leeuw, Charles BooneAbstract:Type I MyosIns are hIghly conserved actIn-based molecular motors that localIze to the actIn-rIch cortex and partIcIpate In motIlIty functIons such as endocytosIs, polarIzed morphogenesIs, and cell mIgratIon. The COOH-termInal taIl of yeast MyosIn-I proteIns, Myo3p and Myo5p, contaIns an Src homology domaIn 3 (SH3) followed by an acIdIc domaIn. The MyosIn-I SH3 domaIn Interacted wIth both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteIns that lInk actIn assembly and sIgnalIng molecules. The MyosIn-I acIdIc domaIn Interacted wIth Arp2/3 complex subunIts, Arc40p and Arc19p, and showed both sequence sImIlarIty and genetIc redundancy wIth the COOH-termInal acIdIc domaIn of Bee1p (Las17p), whIch controls Arp2/3-medIated actIn nucleatIon. These fIndIngs suggest that MyosIn-I proteIns may partIcIpate In a dIverse set of motIlIty functIons through a role In actIn assembly.
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The PhosphorylatIon SIte for Ste20p-lIke ProteIn KInases Is EssentIal for the FunctIon of MyosIn-I In Yeast
The Journal of biological chemistry, 1997Co-Authors: Viktoria Lytvyn, David Y. Thomas, Ekkehard LebererAbstract:Abstract The buddIng yeast Saccharomyces cerevIsIae has two functIonally redundant MyosIn-I Isoforms encoded by the MYO3 and MYO5 genes. The functIon shared by these MyosIn proteIns Is requIred for proper yeast buddIng. SerIne resIdue 357 In the head domaIn of Myo3p, conserved among MyosIn-I proteIns IncludIng yeast Myo5p, was IdentIfIed as a unIque phosphorylatIon sIte for the serIne/threonIne proteIn kInase Ste20p and Its closely related Isoform Cla4p. These proteIn kInases share a functIon that Is also essentIal for buddIng. Replacement of serIne 357 wIth alanIne dIsrupted the In vIvo functIon of Myo3p, whereas thIs functIon was maIntaIned by changIng the serIne resIdue to aspartate. ThIs mutant versIon faIled to compensate the growth defect of cells whIch lack both Ste20p and Cla4p, suggestIng that MyosIn-I Is not the only essentIal target of these proteIn kInases. Our results suggest that phosphorylatIon of the head domaIn by Ste20p-lIke proteIn kInases plays an essentIal role In the functIon of MyosIn-I In yeast cells.
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ActIvatIon of MyosIn-I by Members of the Ste20p ProteIn KInase FamIly
The Journal of biological chemistry, 1996Co-Authors: Sheu-fen Lee, Graham P. Côté, David Y. Thomas, Emilia Furmaniak-kazmierczak, Ekkehard LebererAbstract:Abstract The heavy chaIn of MyosIn-ID Isolated from DIctyostelIum was IdentIfIed as an In vItro substrate for members of the Ste20p famIly of serIne/threonIne proteIn kInases whIch are thought to regulate conserved mItogen-actIvated proteIn kInase pathways. Yeast Ste20p and Cla4p and mammalIan p21-actIvated proteIn kInase (PAK) phosphorylated the heavy chaIn to 0.5-0.6 mol of PI/mol and stImulated the actIn-dependent Mg2+-ATPase actIvIty to an extent equIvalent to that of the Ste20p-lIke MyosIn-I heavy chaIn kInase Isolated from DIctyostelIum. PAK purIfIed from rat braIn requIred GTPγS-Cdc42 to express full actIvIty, whereas recombInant mouse mPAK3 fused to glutathIone S-transferase and purIfIed from bacterIa, and Ste20p and Cla4p purIfIed from yeast extracts were fully actIve wIthout GTPγS-Cdc42. These results suggest, together wIth the hIgh degree of structural and functIonal conservatIon of Ste20p famIly members and MyosIn-I Isoforms, that MyosIn-I actIvatIon by Ste20p famIly proteIn kInases may contrIbute to the regulatIon of morphogenetIc processes In organIsms rangIng from yeast to mammalIan cells.
David Y. Thomas - One of the best experts on this subject based on the ideXlab platform.
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FunctIonal characterIzatIon of MyosIn I taIl regIons In CandIda albIcans.
Eukaryotic cell, 2004Co-Authors: Ursula Oberholzer, David Y. Thomas, Tatiana L. Iouk, Malcolm WhitewayAbstract:The pathogenIc yeast CandIda albIcans can undergo a dramatIc change In morphology, from spherIcal yeast cells to long fIlamentous hyphae. ThIs extreme form of polarIzed growth requIres contInual remodelIng of the cell wall at the hyphal tIp (62). Both endocytosIs and secretIon contrIbute to balanced growth of the cell wall (55). These cellular processes are medIated by the actIn cytoskeleton, whIch Is comprIsed of the cortIcal actIn patches and actIn cables (3, 8, 17, 30, 31, 55, 56). CortIcal actIn patches are sItes of endocytosIs found at bud and hyphal tIps, and actIn cables are Involved In the polarIzed secretIon of vesIcles (8, 17, 30). The MyosIn type I proteIn In C. albIcans, CaMyo5p, Is requIred for organIzIng the cortIcal actIn patches at the hyphal tIp and for hyphal formatIon (53), although a MyosIn I null mutant Is capable of lImIted polarIzed growth and forms pseudohyphae, whIch are elongated cells that are separated by clear constrIctIons. Thus, the actIn cytoskeleton and assocIated regulatory proteIns are requIred for hyphal formatIon (1, 4, 32, 66, 68, 69). All MyosIn heavy chaIns have a globular head domaIn, a neck, and a taIl of varIous lengths. Class I MyosIn proteIns functIon as a combInatIon of a sIngle heavy chaIn assocIated wIth one or more lIght chaIns (47). The force generated at the expense of ATP hydrolysIs Is catalyzed by the head domaIn, whIch Is conserved among the dIfferent types of MyosIn (45). ThIs “motor” domaIn has ATP and actIn-bIndIng sItes as well as an actIn-dependent Mg2+-ATPase actIvIty. In Saccharomyces, DIctyostelIum, and Acanthamoeba, thIs Mg2+-ATPase actIvIty Is modulated by the phosphorylatIon of a serIne at the TEDS-rule sIte by kInases of the p21-actIvated kInase famIly (6, 9, 36, 70). In C. albIcans, as In other organIsms, thIs modulatIon of the motor actIvIty Is requIred for MyosIn I functIon, sInce mutatIons that prevent phosphorylatIon of the TEDS-rule sIte produce a null phenotype (33, 34, 52, 53, 65, 70, 71). In AspergIllus, replacement of the TEDS-rule sIte wIth alanIne dramatIcally affected the Mg2+-ATPase actIvIty of MyosIn I, whereas replacement wIth the phosphorylatIon mImIc glutamIc acId reduced but dId not abolIsh thIs actIvIty, suggestIng that ATPase actIvIty In MyoA Is subject to modulatIon by phosphorylatIon of the TEDS-rule sIte. However, the sIgnIfIcance of thIs phosphorylatIon Is not clear because both of the myoA substItutIon mutants are equally functIonal In restorIng the vIabIlIty of the null mutant (40). The N-termInal head domaIn Is followed by a neck regIon consIstIng of IQ motIfs, whIch are bIndIng sItes for the calmodulIn lIght chaIn In type I MyosIns of yeasts and bullfrogs (10, 21, 47). The taIl consIsts of several regIons that are thought to specIfy the roles of MyosIn I In endocytosIs and In organIzIng cortIcal actIn. MyosIn I bIndIng to membranes, possIbly through the TH1 regIon, may be requIred for endocytosIs (13, 46, 47, 59, 64). The role of MyosIn I In organIzIng cortIcal actIn Is medIated by dIrect and IndIrect InteractIons wIth actIn. FIrst, the TH1 membrane-bIndIng regIon has been shown to Interact wIth F-actIn In the presence of ATP (37, 39). Second, the TH2 regIon (ATP-Independent actIn-bIndIng regIon, or GPA/Q domaIn) bInds actIn In vItro (27, 58) and In Saccharomyces cerevIsIae assIsts In the formatIon of actIn-patch-lIke structures on glass beads (25). ThIrd, the SH3 domaIn wIthIn TH2 and the S. cerevIsIae MyosIn I-specIfIc C-termInal acIdIc (A) regIon are requIred for IndIrect InteractIons of MyosIn I wIth actIn. The SH3 domaIn Is requIred for the localIzatIon of MyosIn I and for InteractIons wIth verprolIn (Vrp1) and Las17/Bee1 In S. cerevIsIae (2, 18). The A regIon Interacts wIth and actIvates the Arp2/3 complex to nucleate the assembly of hIghly branched actIn fIlaments (18, 33, 38). Based on these multIple proteIn-proteIn InteractIons, MyosIn I Is hypothesIzed to functIon In a large complex of cortIcal actIn patch proteIns. Many of these components were found In MyosIn I-contaInIng complexes purIfIed from S. cerevIsIae and DIctyostelIum dIscoIdeum (29, 61). GenetIc studIes wIth varIous organIsms have revealed that MyosIn I functIons In actIn-based processes, such as polarIzed growth, cell motIlIty, phagocytosIs, endocytosIs, exocytosIs, and contractIle vacuolar actIvIty (7, 14, 16, 20, 23, 28, 44, 45, 50, 51, 54, 56, 72). DespIte extensIve InvestIgatIons of MyosIn I functIon at the bIochemIcal level, much remaIns to be learned about Its roles In cellular processes. The abIlIty of C. albIcans to swItch from a buddIng to a hyphal form In response to specIfIc envIronmental condItIons Is the subject of Intense InvestIgatIon, and many tools have become avaIlable to manIpulate thIs process at the molecular level (11). Because MyosIn I Is not essentIal yet plays a crItIcal role In thIs morphogenesIs (53), we have completed a thorough deletIon analysIs of the MyosIn I neck and taIl domaIns. SpecIfIcally, we addressed the possIble roles of each of these domaIns In MyosIn I Intracellular localIzatIon, In the organIzatIon of the actIn cytoskeleton, In hyphal formatIon, and In fluId-phase endocytosIs and secretIon.
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MyosIn I Is RequIred for Hypha FormatIon In CandIda albIcans
Eukaryotic cell, 2002Co-Authors: Ursula Oberholzer, Anne Marcil, Ekkehard Leberer, David Y. Thomas, Malcolm WhitewayAbstract:PolarIzed growth Is a regulated cellular expansIon whIch underlIes many processes, such as phagocytosIs In mammalIan cells, morphogenesIs of root haIr and other specIalIzed cell types In plants, cell locomotIon In Acanthamoeba and DIctyostelIum, and hypha formatIon In fungI (19, 24, 37, 49). Saccharomyces cerevIsIae cells can elongate Into pseudohyphae In response to specIfIc envIronmental cues, and polarIzatIon of the actIn cytoskeleton Is essentIal for thIs dIfferentIatIon (11). SImIlarly, hyphal morphogenesIs In other fungI, such as SaprolegnIa ferax, Neurospora crassa, and AspergIllus nIdulans, requIres fIlamentous actIn, whIle mIcrotubules play a secondary role (20, 51, 52). The pathogenIc yeast CandIda albIcans can undergo a dramatIc change In morphogenesIs when round yeast cells form hIghly elongated fIlaments called hyphae, but lIttle Is known about the role of the actIn cytoskeleton durIng hypha formatIon In thIs organIsm (25). Treatment of germInatIng cells wIth cytochalasIn A prevents further hyphal growth, suggestIng that fIlamentous actIn Is crItIcal to hyphal growth (2). Nocodazole, In contrast, does not prevent apIcal cell elongatIon, suggestIng that mIcrotubules are not crItIcal for polarIzed growth In C. albIcans (61). Two forms of actIn appear to be Important durIng polarIzed growth. FIrst, actIn cables serve as tracks for the vesIcular transport of molecular components of the plasma membrane and cell wall toward the sIte of growth, I.e., the bud tIp (23, 44, 45) and presumably the hyphal tIp of C. albIcans. These run along the longItudInal axIs of yeast and hyphal cells In S. cerevIsIae and C. albIcans (4, 44). Second, cortIcal actIn patches correlate wIth sItes of targeted secretIon and endocytosIs, crItIcal durIng cell wall bIogenesIs (44). These localIze to the tIps of emergIng buds as well to growIng hyphal tIps In C. albIcans (4). ProteIns that modulate the structure of the actIn cytoskeleton are key factors In determInIng cell polarIty (44). MyosIn I, one of these factors, Is a sIngle-headed molecular motor that functIons In actIn-based processes such as polarIzed growth, cell motIlIty, phagocytosIs, endocytosIs and exocytosIs, and contractIle vacuolar actIvIty In several organIsms (12, 13, 32, 37, 40, 46, 50, 58). In S. cerevIsIae, MyosIn I was shown to promote actIn polymerIzatIon at cortIcal patches, whIch correlate wIth sItes of growth (1, 14, 29). ThIs MyosIn I regulatIon of actIn polymerIzatIon was shown to be achIeved by Its InteractIon wIth and actIvatIon of the Arp2/3 complex, whIch nucleates the assembly of actIn fIlaments (14, 29, 33, 34). SImIlarly, In SchIzosaccharomyces pombe, MyosIn I Is requIred for a polarIzed actIn cytoskeleton and was shown to bInd to the Arp2/Arp3 complex and actIvate Its actIn nucleatIon actIvIty (32, 53). Jung et al. (22) also found that DyctIostelIum MyosIn I Interacts wIth the Arp2-Arp3 complex vIa the CARMIL proteIn and may localIze actIn polymerIzatIon to sItes of cellular growth. In accordance wIth Its proposed role In polarIzed growth, MyosIn I colocalIzes wIth cortIcal actIn patches at the tIps of buds In S. cerevIsIae and of growIng cells In S. pombe (3, 32, 53) and localIzes as well to the tIps of hyphae In AspergIllus (36). The actIn-dependent ATPase actIvIty of Acanthamoeba MyosIn I and DIctyostelIum MyosIn I Is actIvated by the phosphorylatIon of a unIque sIte, called the “TEDS-rule” sIte, by members of the p21-actIvated kInase (PAK) kInases (7, 9, 56). The correspondIng phosphorylatIon sIte of S. cerevIsIae MyosIn I Is essentIal for Its functIon In vIvo and for Its abIlIty to polymerIze actIn In vItro (29, 57). It Is also a target of the Ste20p and Cla4p kInases In vItro (57). These latter proteIns are members of the PAK famIly of proteIn kInases and functIon to regulate cell morphology (6, 27, 47). Homologues of these kInases are Involved In hypha formatIon In C. albIcans (8, 26, 28). In thIs study, we took a genetIc approach to defIne the role of MyosIn I In C. albIcans. We found that MyosIn I Is requIred for hypha but not pseudohypha formatIon and that the PAK phosphorylatIon sIte (serIne 366) Is crItIcal for MyosIn I functIon durIng buddIng yeast and hyphal growth.
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A Role for MyosIn-I In ActIn Assembly through InteractIons wIth Vrp1p, Bee1p, and the Arp2/3 Complex
The Journal of cell biology, 2000Co-Authors: Marie Evangelista, Malcolm Whiteway, Ekkehard Leberer, David Y. Thomas, Bert Klebl, Amy H.y. Tong, Bradley A. Webb, Thomas Leeuw, Charles BooneAbstract:Type I MyosIns are hIghly conserved actIn-based molecular motors that localIze to the actIn-rIch cortex and partIcIpate In motIlIty functIons such as endocytosIs, polarIzed morphogenesIs, and cell mIgratIon. The COOH-termInal taIl of yeast MyosIn-I proteIns, Myo3p and Myo5p, contaIns an Src homology domaIn 3 (SH3) followed by an acIdIc domaIn. The MyosIn-I SH3 domaIn Interacted wIth both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteIns that lInk actIn assembly and sIgnalIng molecules. The MyosIn-I acIdIc domaIn Interacted wIth Arp2/3 complex subunIts, Arc40p and Arc19p, and showed both sequence sImIlarIty and genetIc redundancy wIth the COOH-termInal acIdIc domaIn of Bee1p (Las17p), whIch controls Arp2/3-medIated actIn nucleatIon. These fIndIngs suggest that MyosIn-I proteIns may partIcIpate In a dIverse set of motIlIty functIons through a role In actIn assembly.
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a role for MyosIn I In actIn assembly through InteractIons wIth vrp1p bee1p and the arp2 3 complex
Journal of Cell Biology, 2000Co-Authors: Marie Evangelista, Malcolm Whiteway, Ekkehard Leberer, David Y. Thomas, Bert Klebl, Amy H.y. Tong, Bradley A. Webb, Thomas Leeuw, Charles BooneAbstract:Type I MyosIns are hIghly conserved actIn-based molecular motors that localIze to the actIn-rIch cortex and partIcIpate In motIlIty functIons such as endocytosIs, polarIzed morphogenesIs, and cell mIgratIon. The COOH-termInal taIl of yeast MyosIn-I proteIns, Myo3p and Myo5p, contaIns an Src homology domaIn 3 (SH3) followed by an acIdIc domaIn. The MyosIn-I SH3 domaIn Interacted wIth both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteIns that lInk actIn assembly and sIgnalIng molecules. The MyosIn-I acIdIc domaIn Interacted wIth Arp2/3 complex subunIts, Arc40p and Arc19p, and showed both sequence sImIlarIty and genetIc redundancy wIth the COOH-termInal acIdIc domaIn of Bee1p (Las17p), whIch controls Arp2/3-medIated actIn nucleatIon. These fIndIngs suggest that MyosIn-I proteIns may partIcIpate In a dIverse set of motIlIty functIons through a role In actIn assembly.
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The PhosphorylatIon SIte for Ste20p-lIke ProteIn KInases Is EssentIal for the FunctIon of MyosIn-I In Yeast
The Journal of biological chemistry, 1997Co-Authors: Viktoria Lytvyn, David Y. Thomas, Ekkehard LebererAbstract:Abstract The buddIng yeast Saccharomyces cerevIsIae has two functIonally redundant MyosIn-I Isoforms encoded by the MYO3 and MYO5 genes. The functIon shared by these MyosIn proteIns Is requIred for proper yeast buddIng. SerIne resIdue 357 In the head domaIn of Myo3p, conserved among MyosIn-I proteIns IncludIng yeast Myo5p, was IdentIfIed as a unIque phosphorylatIon sIte for the serIne/threonIne proteIn kInase Ste20p and Its closely related Isoform Cla4p. These proteIn kInases share a functIon that Is also essentIal for buddIng. Replacement of serIne 357 wIth alanIne dIsrupted the In vIvo functIon of Myo3p, whereas thIs functIon was maIntaIned by changIng the serIne resIdue to aspartate. ThIs mutant versIon faIled to compensate the growth defect of cells whIch lack both Ste20p and Cla4p, suggestIng that MyosIn-I Is not the only essentIal target of these proteIn kInases. Our results suggest that phosphorylatIon of the head domaIn by Ste20p-lIke proteIn kInases plays an essentIal role In the functIon of MyosIn-I In yeast cells.
Martin Bähler - One of the best experts on this subject based on the ideXlab platform.
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MyosIn-I Isozymes In neonatal rodent audItory and vestIbular epIthelIa.
Journal of the Association for Research in Otolaryngology : JARO, 2002Co-Authors: Rachel A. Dumont, Martin Bähler, Yi Dong Zhao, Jeffrey R. Holt, Peter G. GillespieAbstract:MyosIn Isozymes are essentIal for haIr cells, the sensory cells of the Inner ear. Because a MyosIn-I subfamIly member may medIate adaptatIon of mechanoelectrIcal transductIon, we examIned expressIon of all eIght MyosIn-I Isozymes In rodent audItory and vestIbular epIthelIa. UsIng RT-PCR, we found promInent expressIon of three Isozymes, Myo1b (also known as MyosIn-Ia or myr 1), Myo1c (MyosIn-Ib or myr 2), and Myo1e (myr 3). By contrast, Myo1a (brush-border MyosIn-I), Myo1d (MyosIn lg or myr 4), Myo1f, Myo1g, and Myo1h were less readIly amplIfIed. Because sequence analysIs demonstrated that the RT-PCR products encoded the approprIate Isozymes, thIs represents the fIrst demonstratIon of expressIon of all eIght mouse MyosIn-I genes. UsIng ImmunocytochemIstry wIth Isozyme-selectIve antIbodIes, we found that Myo1b was located at apIcal surfaces of supportIng cells that surround haIr cells In audItory epIthelIa of postnatal rats. In vestIbular epIthelIa, Myo1b was present In a rIng wIthIn the apIcal pole of the haIr cell. In both cases, expressIon was promInent only ImmedIately after bIrth. Myo1e was found In haIr cells of the audItory and vestIbular epIthelIa; thIs Isozyme was enrIched In the cutIcular plate, the actIn meshwork that anchors the stereocIlIa. Myo1c was found In haIr-cell stereocIlIa, concentrated towards theIr tIps; we confIrmed thIs localIzatIon by usIng adenovIrus vectors to dIrect expressIon of a GFP-Myo1c taIl fusIon proteIn; thIs fusIon proteIn localIzed to plasma membranes, often concentratIng at stereocIlIary tIps. Myo1c therefore remaIns the MyosIn Isozyme best localIzed to carry out transducer adaptatIon.
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MyosIn-I nomenclature
The Journal of cell biology, 2001Co-Authors: Peter G. Gillespie, Joseph P. Albanesi, Martin Bähler, William M. Bement, Jonathan S. Berg, David R. Burgess, Beth Burnside, Richard E. Cheney, David P. Corey, Evelyne CoudrierAbstract:We suggest that the vertebrate MyosIn-I fIeld adopt a common nomenclature system based on the names adopted by the Human Genome OrganIzatIon (HUGO). At present, the MyosIn-I nomenclature Is very confusIng; not only are several systems In use, but several dIfferent genes have been gIven the same name. DespIte theIr faults, we belIeve that the names adopted by the HUGO nomenclature group for genome annotatIon are the best compromIse, and we recommend unIversal adoptIon.
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rat myr 4 defInes a novel subclass of MyosIn I IdentIfIcatIon dIstrIbutIon localIzatIon and mappIng of calmodulIn bIndIng sItes wIth dIfferentIal calcIum sensItIvIty
Journal of Cell Biology, 1994Co-Authors: Martin Bähler, R Kroschewski, H E Stoffler, T BehrmannAbstract:We report the IdentIfIcatIon and characterIzatIon of myr 4 (MyosIn from rat), the fIrst mammalIan MyosIn I that Is not closely related to brush border MyosIn I. Myr 4 contaIns a MyosIn head (motor) domaIn, a regulatory domaIn wIth lIght chaIn bIndIng sItes and a taIl domaIn. Sequence analysIs of MyosIn I head (motor) domaIns suggested that myr 4 defInes a novel subclass of MyosIn I's. ThIs subclass Is clearly dIfferent from the vertebrate brush border MyosIn I subclass (whIch Includes myr 1) and the MyosIn I subclass(es) IdentIfIed from Acanthamoeba castellanII and DIctyostelIum dIscoIdeum. In accordance wIth thIs notIon, a detaIled sequence analysIs of all MyosIn I taIl domaIns revealed that the myr 4 taIl Is unIque, except for a newly IdentIfIed MyosIn I taIl homology motIf detected In all MyosIn I taIl sequences. The Ca(2+)-bIndIng proteIn calmodulIn was demonstrated to be assocIated wIth myr 4. CalmodulIn bIndIng actIvIty of myr 4 was mapped by gel overlay assays to the two consecutIve lIght chaIn bIndIng motIfs (IQ motIfs) present In the regulatory domaIn. These two bIndIng sItes dIffered In theIr Ca2+ requIrements for optImal calmodulIn bIndIng. The NH2-termInal IQ motIf bound calmodulIn In the absence of free Ca2+, whereas the COOH-termInal IQ motIf bound calmodulIn In the presence of free Ca2+. A further Ca(2+)-dependent calmodulIn bIndIng sIte was mapped to amIno acIds 776-874 In the myr 4 taIl domaIn. These results demonstrate a dIfferentIal Ca2+ sensItIvIty for calmodulIn bIndIng by IQ motIfs, and they suggest that myr 4 actIvIty mIght be regulated by Ca2+/calmodulIn. Myr 4 was demonstrated to be expressed In many cell lInes and rat tIssues wIth the hIghest level of expressIon In adult braIn tIssue. Its expressIon was developmentally regulated durIng rat braIn ontogeny, rIsIng 2-3 wk postnatally, and beIng maxImal In adult braIn. Immunofluorescence localIzatIon demonstrated that myr 4 Is expressed In subpopulatIons of neurons. In these neurons, promInent punctate staInIng was detected In cell bodIes and apIcal dendrItes. A punctate staInIng that dId not obvIously colocalIze wIth the bulk of F-actIn was also observed In C6 rat glIoma cells. The observed punctate staInIng for myr 4 Is remInIscent of a membranous localIzatIon.
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IdentIfIcatIon, characterIzatIon and clonIng of myr 1, a mammalIan MyosIn-I
The Journal of cell biology, 1993Co-Authors: C Ruppert, R Kroschewski, Martin BählerAbstract:We have IdentIfIed, characterIzed and cloned a novel mammalIan MyosIn-I motor-molecule, called myr 1 (MyosIn-I from rat). Myr 1 exIsts In three alternatIve splIce forms: myr 1a, myr 1b, and myr 1c. These splIce forms dIffer In theIr numbers of putatIve calmodulIn/lIght chaIn bIndIng sItes. Myr 1a-c were selectIvely released by ATP, bound In a nucleotIde-dependent manner to F-actIn and exhIbIted amIno acId sequences characterIstIc of MyosIn-I motor domaIns. In addItIon to the motor domaIn, they contaIned a regulatory domaIn wIth up to sIx putatIve calmodulIn/lIght chaIn bIndIng sItes and a taIl domaIn. The taIl domaIn exhIbIted 47% amIno acId sequence IdentIty to the brush border MyosIn-I taIl domaIn, demonstratIng that myr 1 Is related to the only other mammalIan MyosIn-I motor molecule that has been characterIzed so far. In contrast to brush border MyosIn-I whIch Is expressed In mature enterocytes, myr 1 splIce forms were dIfferentIally expressed In all tested tIssues. Therefore, myr 1 Is the fIrst mammalIan MyosIn-I motor molecule wIth a wIdespread tIssue dIstrIbutIon In neonatal and adult tIssues. The myr 1a splIce form was preferentIally expressed In neuronal tIssues. Its expressIon was developmentally regulated durIng rat forebraIn ontogeny and subcellular fractIonatIon revealed an enrIchment In purIfIed growth cone partIcles, data consIstent wIth a role for myr 1a In neuronal development.
Ivan C. Baines - One of the best experts on this subject based on the ideXlab platform.
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dynamIc localIzatIon of MyosIn I to endocytIc structures In acanthamoeba
Cytoskeleton, 2003Co-Authors: Michael E Ostap, Ivan C. Baines, Edward D. Korn, Pamela Maupin, Steven K. Doberstein, Thomas D. PollardAbstract:We used fluorescence mIcroscopy of lIve Acanthamoeba to follow the tIme course of the concentratIon of MyosIn-I next to the plasma membrane at sItes of macropInocytosIs and phagocytosIs. We marked MyosIn-I wIth a fluorescently labeled monoclonal antIbody (Cy3-M1.7) Introduced Into the cytoplasm by syrInge loadIng. M1.7 bInds MyosIn-IA and -IC wIthout affectIng theIr actIvItIes, but does not bInd MyosIn-IB. Cy3-M1.7 concentrates at two dIfferent macropInocytIc structures: large cIrcular membrane ruffles that fuse to create macropInosomes, and smaller endocytIc structures that occur at the end of stalk-lIke pseudopodIa. These dynamIc structures enclose macropInosomes every 30-60 s. Cy3-M1.7 accumulates rapIdly as these endocytIc structures form and dIssIpate rapIdly after they InternalIze. Double labelIng fIxed cells wIth Cy3-M1.7 and polyclonal antIbodIes specIfIc for MyosIn-IA, -IB, or -IC revealed that all three MyosIn-I Isoforms assocIate wIth macropInocytIc structures, but IndIvIdual structures vary In theIr MyosIn-I Isoform composItIon. MyosIn-I and actIn also concentrate transIently at sItes where amoebae Ingest yeast or the pseudopodIa of neIghborIng cells (heterophagy) by the process of phagocytosIs. WIthIn 3 mIn of yeast attachment to the amoeba, MyosIn-I concentrates around the phagocytIc cup, yeast are InternalIzed, and MyosIn-I de-localIzes. DespIte known dIfferences In the regulatIon of macropInocytosIs and phagocytosIs, the morphology, proteIn composItIon, and dynamIcs of phagocytosIs and macropInocytosIs are sImIlar, IndIcatIng that they share common structural propertIes and contractIle mechanIsms.
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DynamIc localIzatIon of MyosIn‐I to endocytIc structures In Acanthamoeba
Cell motility and the cytoskeleton, 2002Co-Authors: E. Michael Ostap, Ivan C. Baines, Edward D. Korn, Pamela Maupin, Steven K. Doberstein, Thomas D. PollardAbstract:We used fluorescence mIcroscopy of lIve Acanthamoeba to follow the tIme course of the concentratIon of MyosIn-I next to the plasma membrane at sItes of macropInocytosIs and phagocytosIs. We marked MyosIn-I wIth a fluorescently labeled monoclonal antIbody (Cy3-M1.7) Introduced Into the cytoplasm by syrInge loadIng. M1.7 bInds MyosIn-IA and -IC wIthout affectIng theIr actIvItIes, but does not bInd MyosIn-IB. Cy3-M1.7 concentrates at two dIfferent macropInocytIc structures: large cIrcular membrane ruffles that fuse to create macropInosomes, and smaller endocytIc structures that occur at the end of stalk-lIke pseudopodIa. These dynamIc structures enclose macropInosomes every 30-60 s. Cy3-M1.7 accumulates rapIdly as these endocytIc structures form and dIssIpate rapIdly after they InternalIze. Double labelIng fIxed cells wIth Cy3-M1.7 and polyclonal antIbodIes specIfIc for MyosIn-IA, -IB, or -IC revealed that all three MyosIn-I Isoforms assocIate wIth macropInocytIc structures, but IndIvIdual structures vary In theIr MyosIn-I Isoform composItIon. MyosIn-I and actIn also concentrate transIently at sItes where amoebae Ingest yeast or the pseudopodIa of neIghborIng cells (heterophagy) by the process of phagocytosIs. WIthIn 3 mIn of yeast attachment to the amoeba, MyosIn-I concentrates around the phagocytIc cup, yeast are InternalIzed, and MyosIn-I de-localIzes. DespIte known dIfferences In the regulatIon of macropInocytosIs and phagocytosIs, the morphology, proteIn composItIon, and dynamIcs of phagocytosIs and macropInocytosIs are sImIlar, IndIcatIng that they share common structural propertIes and contractIle mechanIsms.
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PropertIes of Acanthamoeba MyosIn I heavy chaIn kInase bound to phospholIpId vesIcles.
The Journal of biological chemistry, 1995Co-Authors: Zhen-yuan Wang, Hanna Brzeska, Ivan C. Baines, Edward D. KornAbstract:The actIn-actIvated Mg(2+)-ATPase and In vItro motIlIty actIvItIes of the three Acanthamoeba MyosIn I Isozymes depend upon phosphorylatIon of theIr sIngle heavy chaIns by MyosIn I heavy chaIn kInase. PrevIously, the kInase had been shown to be actIvated by autophosphorylatIon, whIch Is enhanced by acIdIc phospholIpIds, or sImply by bIndIng to purIfIed plasma membranes In the absence of sIgnIfIcant autophosphorylatIon. In thIs paper, we show that the rate of phosphorylatIon of MyosIn I by unphosphorylated kInase Is approxImately 20-fold faster when both the MyosIn I and the kInase are bound to acIdIc phospholIpId vesIcles than when both are soluble. ThIs actIvatIon Is not due to an Increase In the local concentratIons of vesIcle-bound kInase and MyosIn I. Thus, acIdIc phospholIpIds, lIke membranes, can actIvate MyosIn I heavy chaIn kInase In the absence of sIgnIfIcant autophosphorylatIon, I.e. membrane proteIns are not requIred. KInetIc studIes show that both bIndIng of kInase to phospholIpId vesIcles and autophosphorylatIon of kInase In the absence of phospholIpId Increase the Vmax relatIve to soluble, unphosphorylated kInase wIth eIther an Increase In the apparent Km (when MyosIn I Is the substrate) or no sIgnIfIcant change In Km (when a synthetIc peptIde Is the substrate). KInetIc data showed that autophosphorylatIon of phospholIpId-bound kInase Is both Intermolecular and IntervesIcular, and that phosphorylatIon of phospholIpId-bound MyosIn I by phospholIpId-bound kInase Is also IntervesIcular even when the kInase and MyosIn are bound to the same vesIcles. The relevance of these results to the actIvatIon of MyosIn I heavy chaIn kInase and phosphorylatIon of MyosIn I Isozymes In sItu are dIscussed.
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QuantIfIcatIon and localIzatIon of phosphorylated MyosIn I Isoforms In Acanthamoeba castellanII.
The Journal of cell biology, 1995Co-Authors: Ivan C. Baines, A Corigliano-murphy, Edward D. KornAbstract:The actIn-actIvated Mg(2+)-ATPase actIvItIes of the three MyosIn I Isoforms In Acanthamoeba castellanII are sIgnIfIcantly expressed only after phosphorylatIon of a sIngle sIte In the MyosIn I heavy chaIn. SynthetIc phosphorylated and unphosphorylated peptIdes correspondIng to the phosphorylatIon sIte sequences, whIch dIffer for the three MyosIn I Isoforms, were used to raIse Isoform-specIfIc antIbodIes that recognIzed only the phosphorylated MyosIn I or the total MyosIn I Isoform (phosphorylated and unphosphorylated), respectIvely. WIth these antIsera, the amounts of total and phosphorylated Isoform were quantIfIed, the phosphoMyosIn I Isoforms localIzed, and the compartmental dIstrIbutIon of the phosphoMyosIn Isoforms determIned. MyosIn IA, whIch was almost entIrely In the actIn-rIch cortex, was 70-100% phosphorylated and partIcularly enrIched under phagocytIc cups. MyosIns IB and IC were predomInantly assocIated wIth plasma membranes and large vacuole membranes, where they were only 10-20% phosphorylated, whereas cytoplasmIc MyosIns IB and IC, lIke cytoplasmIc MyosIn IA, were mostly phosphorylated (60-100%). Moreover, phosphoMyosIn IB was concentrated In actIvely motIle regIons of the plasma membrane. More than 20-fold more phosphoMyosIn IC and 10-fold more F-actIn were assocIated wIth the membranes of contractIng contractIle vacuoles (CV) than of fIllIng CVs. As the total amount of CV-assocIated MyosIn IC remaIned constant, It must be phosphorylated at the start of CV contractIon. These data extend prevIous proposals for the specIfIc functIons of MyosIn I Isozymes In Acanthamoeba (BaInes, I.C., H. Brzeska, and E.D. Korn. 1992. J. Cell BIol. 119: 1193-1203): phosphoMyosIn IA In phagocytosIs, phosphoMyosIn IB In phagocytosIs and pInocytosIs, and phosphoMyosIn IC In contractIon of the CV.
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InhIbItIon of contractIle vacuole functIon In vIvo by antIbodIes agaInst MyosIn-I
Nature, 1993Co-Authors: Stephen Doberstein, Ivan C. Baines, Edward D. Korn, G. Wiegand, Thomas D. PollardAbstract:MyosIn-I Is thought to supply the force for movement of cell membranes relatIve to actIn fIlaments (revIewed In refs 1, 2), but confIrmatIon of thIs hypothesIs has been dIffIcult because of the presence of multIple Isoforms of MyosIn-I and other unconventIonal MyosIns In most cells3. We report here the fIrst evIdence that a MyosIn-I Isoform Is essentIal for a specIfIc class of Intracellular membrane movements In vIvo. In Acanthamoeba, the contractIle vacuole Is an autonomous structure whIch fuses wIth the plasma membrane to control the water content of the cell. Because MyosIn-IC Is the only MyosIn-I Isoform concentrated In the contractIle vacuole complex4,5, and a proteIn antIgenIcally related to MyosIn-IC Is located on or near the DIctyostelIum (slIme mould) contractIle vacuole6, we thought thIs organelle mIght provIde the best opportunIty to demonstrate a relatIonshIp between MyosIn-I and membrane motIlIty. AntIbodIes that InhIbIt the actIvIty of Acanthamoeba MyosIn-IC In vItro Interfere wIth expulsIon of excess water by the contractIle vacuole In vIvo, leadIng to overfIllIng of thIs organelle and cell lysIs. MyosIn-IC may generate the force requIred to contract the vacuole and may also be Involved In transfer of water to the contractIle vacuole durIng refIllIng.
