Yes / The individual molecular pathways downstream of Cdc42, Rac, and Rho GTPases are well documented, but we know surprisingly little about how these pathways are coordinated when cells move in a complex environment in vivo. In the developing embryo, melanoblasts originating from the neural crest must traverse the dermis to reach the epidermis of the skin and hair follicles. We previously established that Rac1 signals via Scar/WAVE and Arp2/3 to effect pseudopod extension and migration of melanoblasts in skin. Here we show that RhoA is redundant in the melanocyte lineage but that Cdc42 coordinates multiple motility systems independent of Rac1. Similar to Rac1 knockouts, Cdc42 null mice displayed a severe loss of pigmentation, and melanoblasts showed cell-cycle progression, migration, and cytokinesis defects. However, unlike Rac1 knockouts, Cdc42 null melanoblasts were elongated and displayed large, bulky pseudopods with dynamic actin bursts. Despite assuming an elongated shape usually associated with fast mesenchymal motility, Cdc42 knockout melanoblasts migrated slowly and inefficiently in the epidermis, with nearly static pseudopods. Although much of the basic actin machinery was intact, Cdc42 null cells lacked the ability to polarize their Golgi and coordinate motility systems for efficient movement. Loss of Cdc42 de-coupled three main systems: actin assembly via the formin FMNL2 and Arp2/3, active myosin-II localization, and integrin-based adhesion dynamics. / Cancer Research UK (to L.M.M. [A17196], R.H.I. [A19257], and S.W.G.T.) and NIH grants P01-GM103723 and P41-EB002025 (to K.M.H.). N.R.P. is supported by a Pancreatic Cancer Research Fund grant (to L.M.M.). Funding to Prof. Rottner by the Deutsche Forschungsgemeinschaft (grant RO2414/3-2).

Le trafic intracellulaire consiste en la formation et le transport de vésicules ou tubules qui acheminent des composants protéiques et lipidiques entre les différents organites ou avec la membrane plasmique. L’élaboration de ces tubulo-vésicules est initiée par le remodelage local d’une membrane, tout d’abord en générant une courbure puis un bourgeon qui, s’allongeant, forme la tubulo-vésicule. Enfin, la rupture de la membrane, ou scission, libère le transporteur nouvellement formé. Ces étapes repose sur un sculptage profond de la membrane. Ceci requière des forces générées par des moteurs moléculaires, lesquels s’associent aux cytosquelettes comme les microtubules ou les filaments d’actine. Afin de mieux comprendre comment le cytosquelette et leurs moteurs façonnent ces transporteurs, nous avons examiné le rôle de l’actine et de la myosine VI dans la formation de tubules membranaires aux mélanosomes. Les mélanosomes sont des organites apparentés aux lysosomes, générés dans les mélanocytes de la peau et de la choroïde de l’œil, et qui sont le lieu de synthèse et de stockage d’un pigment, la mélanine. Dans l’épiderme, ces compartiments spécialisés évoluent par différentes étapes de maturation qui aboutissent à leurs transferts aux cellules voisines, les kératinocytes. Les mélanosomes sont des organites dynamiques qui reçoivent et recyclent constamment des composants membranaires, comme la SNARE VAMP7. Nous résultats montrent que la myosine VI et son adapteur optineurine se localisent à un sous-domaine spécifique de la membrane des mélanosomes, ou elles contrôlent la scission de tubules. En effet, l’activité motrice de la myosine VI et le réseau d’actine branchée, dépendant des complexes Arp2/3 et WASH, permettent la constriction des membranes du tubule et son détachement du mélanosome. Un défaut de scission de ces tubes engendre des mélanosomes plus pigmentés, enrichis en cargos et au pH plus acide. L’altération de l’homéostasie du mélanosome affecte sa fonction, comme sa capacité à être sécrété et transféré aux kératinocytes. Nos résultats démontrent que la myosine VI en coopération avec le cytosquelette d’actine permet la constriction et fission de membranes aux mélanosomes. Les intermédiaires de transport ainsi formés recyclent des protéines cargos pour leur possible réutilisation, et participent ainsi au maintien de l’homéostasie et de la fonction de ces organites. / Intracellular transport among organelles and the plasma membrane occurs through the formation and transport of vesicular and tubular membrane carriers. The formation of these carriers requires first the bending of membrane and the generation of a bud, followed by its elongation to form the tubule-vesicle. Lastly, the carrier is released from the membrane source by the scission of the membrane. Importantly, all these different steps need an accurate orchestration to properly deform the membrane. The actions exerted by molecular motors onto microtubule and actin cytoskeletons provide forces onto membrane that contribute to its remodeling during the biogenesis of carrier. Actin filaments (F-actin) and myosins are thought to participate in the initiation and the fission of carriers. However, the role of actin machinery during carrier biogenesis remains elusive. We thus decided to address the role of F-actin and the actin-based motor myosin VI in the formation of tubular intermediates at melanosome. Melanosomes are lysosome-related organelles of skin melanocytes and eye pigment cells that function in the synthesis and storage of the melanin pigment. Melanosomes originate from endosomes and progressively mature into fully pigmented compartments, which fate is to be secreted and transferred to neighboring keratinocytes. Melanosomes are dynamic organelles that constantly receive, but also recycle proteins such as the SNARE VAMP7 through the formation and release of tubular intermediates. Our work reveals that myosin VI, together with Arp2/3- and WASH-mediated branched actin localize at specific melanosomal subdomains where they promote the constriction and scission of tubular intermediates. This fission event allows the export of components such as VAMP7 from melanosomes and promotes their maturation and subsequent transfer to keratinocytes. Altogether, our results uncover a new role for myosin VI and F-actin in the constriction and scission of membrane tubules at melanosome that is required for organelle homeostasis and function.

Animal morphogenesis is largely driven by concerted changes in the shape of individual cells. However, how cell shape changes are regulated and coordinated in developing animals is not well understood. Here we show that the two perpendicular signaling gradients of the morphogens Dpp, a TGF-β homologue, and Wingless, a Wnt family member, maintain tissue homoeostasis and control cell shape changes in the developing Drosophila wing. Clones of cells lacking Dpp or Wingless signaling invaginate apically, shorten apico-basally and subsequently extrude basally without disruption of the epithelium. During early larval development, the onset of Dpp and Wingless signaling correlates with the cuboidal-to-columnar cell shape transition of wing disc cells. Gradients in apical-basal length of columnar cells correlate during late larval development with the gradients of Dpp and Wingless signaling activities. Cells receiving high levels of Dpp and Wingless signaling are most elongated and apically constricted. Low levels of Dpp and Wingless signaling correlate with a shorter and apically wider cell morphology. Dpp and Wingless signaling is cell-autonomously required for maintaining the elongated columnar cell shape of late larval wing disc cells. Overactivation of these pathways results in precocious cell elongation during early larval development. These morphogenetic responses to Dpp and Wingless require the transcription factor complexes Mad and Tcf/β-catenin, respectively, indicating that they are mediated by changes in gene expression. The morphogenetic function of Wingless is in part mediated by one of its target genes, the transcription factor Vestigial. Wingless signaling promotes an enrichment of E-cadherin at the adherens junctions, and we show that E-cadherin is required to maintain apical-basal cell length. Dpp signaling controls the subcellular distribution of the activities of the small GTPase Rho1 and the regulatory light chain of non-muscle myosin II (MRLC). Alteration of Rho1 or MRLC activity has a profound effect on apical-basal cell length. Finally, we demonstrate that a decrease in Rho1 or MRLC activity rescues the shortening of cells with compromised Dpp signaling. Our results identify cell-autonomous roles for Dpp and Wingless signaling in promoting and maintaining the elongated columnar shape of wing disc cells. Furthermore, they suggest that Dpp and Wingless signaling control cell shape by regulating the actin-MyosinII/E-cadherin network. / Morphogenese in Tieren wird in hohem Maße von konzertierten Zellformveränderungen einzelner Zellen bewirkt. Es ist jedoch noch nicht hinreichend verstanden, wie Zellformveränderungen in sich entwickelnden Tieren reguliert und koordiniert werden. Hier zeigen wir, dass die zwei zueinander senkrecht stehenden Signalgradienten der Morphogene Dpp, eines TGF-β Homologs, und Wingless, eines Mitglieds der Wnt Familie, im sich entwickelnden Drosophila-Flügel Gewebe-Homöostase aufrechterhalten und Zellformveränderungen kontrollieren. Klone von Zellen, denen Dpp oder Wingless Signalaktivität fehlt, invaginieren von ihrer apikalen Seite her, verkürzen sich in apiko-basaler Richtung und extruieren im Folgenden auf der basalen Seite des Epithels, ohne es zu zerstören. Während der frühen Larvalentwicklung korreliert das Anschalten der Dpp und Wingless Signale mit der Zellformveränderung der Flügelscheibenzellen von kuboidal zu kolumnar. Gradienten in der apiko-basalen Länge von kolumnaren Zellen korrelieren während der späten Larvalentwicklung mit den Gradienten der Dpp und Wingless Signalaktivitäten. Zellen, die hohe Werte an Dpp und Wingless Signalen empfangen, sind am meisten elongiert und apikal konstringiert. Niedrige Werte von Dpp und Wingless Signalen korrelieren mit kürzerer und apikal weiterer Zellmorphologie. Dpp und Wingless Signale werden zellautonom gebraucht für die Aufrechterhaltung der elongierten Zellform von späten larvalen Flügelscheibenzellen. Die Überaktivierung dieser Signalwege führt zu vorzeitiger Zellverlängerung während der frühen Larvalentwicklung. Diese morphogenetischen Antworten auf Dpp und Wingless benötigen die Transkriptionsfaktor-Komplexe Mad beziehungsweise Tcf/β-catenin, was darauf hindeutet, dass sie durch Änderungen in der Genexpression vermittelt werden. Die morphogenetische Funktion von Wingless wird teilweise durch eines seiner Zielgene, Vestigial, vermittelt. Wingless Signale fördern die Anreicherung von E-cadherin an den Adherensverbindungen. Wir zeigen hier, dass E-cadherin gebraucht wird, um apiko-basale Zelllänge aufrechtzuerhalten. Dpp Signale kontrollieren die subzelluläre Verteilung der Aktivitäten der kleinen GTPase Rho1 und der regulatorischen leichten Kette von nicht-muskulärem Myosin II (MRLC). Eine Änderung in der Rho1 oder MRLC Aktivität hat weitreichende Auswirkungen auf die apiko-basale Zelllänge. Schließlich zeigen wir noch, dass eine Verringerung der Rho1 oder MRLC Aktivitäten die Zellverkürzung von Dpp-Signal kompromittierten Zellen rettet. Unsere Resultate identifizieren zellautonome Rollen für Dpp und Wingless Signale in der Förderung und Aufrechterhaltung der elongierten kolumnaren Zellform von Flügelimaginalscheibenzellen. Darüber hinaus suggerieren sie, dass Dpp und Wingless Signale die Zellform durch die Regulierung des Aktin-MyosinII/E-cadherin-Netzwerks kontrollieren.

Dissertation (Ph.D.)--University of South Florida, 2008. / Includes vita. Includes bibliographical references (leaves 102-128).

No description available.

Dissertation (Ph.D.)--University of South Florida, 2008. / Title from PDF of title page. Document formatted into pages; contains 132 pages. Includes vita. Includes bibliographical references.

La cytokine TRAIL est un candidat anticancéreux qui induit la mort spécifique de cellules tumorales. La liaison de TRAIL à ses récepteurs (DR) permet de former le complexe DISC qui induit la mort cellulaire. La raison de la mort sélective des cellules tumorales induite par TRAIL est inconnue. Nous avons découvert des partenaires de DR: chaînes lourdes de myosine IIa, IIb (NMHCIIa, NMHCIIb), chaîne légère régulatrice de myosine (MLC2) et ß-actine. Dans les cellules tumorales, la liaison de TRAIL abroge l'interaction NMHCII/DR, et DISC est activé. Au contraire, dans les cellules normales, l'interaction NMHCII/DR persiste et l'activation de DISC est incomplète. Affaiblir l'interaction NMHCII/DR par des inhibiteurs chimiques ou diminuer NMHCIIa permet d'augmenter l'apoptose liée à TRAIL. L'interaction réduite NMHCII/DR induit des niveaux altérés de phospho-MLC2 et de kinases régulant MLC2. Nous proposons que la résistance de cellules normales à TRAIL soit basée sur l'interaction DR/cytosquelette, déficiente dans des tumeurs. NMHCII étant aussi impliqué dans l'adhésion/migration cellulaire, il serait intéressant d'étudier les fonctions de NMHCII/DISC dans le détachement cellulaire, afin de mieux comprendre la résistance à TRAIL de certains cancers. / The cytokine TRAIL is a promising cancer therapeutic candidate as it induces apoptosis selectively in transformed cells. TRAIL-induced clustering of its receptors (DR) is essential for the DISC complex formation, which induces cell death. The mechanism for TRAIL’s tumour selective effect is largely unknown. We identified the cytoskeleton proteins non-muscle myosin heavy chain IIa, IIb (NMHCIIa, NMHCIIb), myosin regulatory light chain (MLC2) and ß-actin as novel DR-interactors. An initially weak and TRAIL-induced abrogation of NMHCII/DR interaction correlated with efficient DISC formation in tumour cells. In contrast, a robust NMHCII/DR interaction that was sustained upon TRAIL stimulus was accompanied by incomplete DISC arrangement. Weakening the NMHCII/DR interaction in normal cells using chemical inhibitors enhanced TRAIL-induced apoptosis. Intriguingly, siRNA-mediated NMHCIIa- but not NMHCIIb depletion potently released TRAIL resistance in normal cells and influenced DISC composition. Reduced NMHCII/DR interaction in transformed cells was characterised by diminished MLC2 phosphorylation and altered protein expression of upstream regulatory kinases. Our results suggest that normal cell resistance to TRAIL-apoptosis is based on the interaction of cytoskeleton components with DR that is impaired upon transformation. Since NMHCII function in cell adhesion and migration, it will be interesting to study possible roles of the interaction in cell detachment and altered TRAIL sensitivity; moreover this link may provide clues as to the cause of TRAIL resistance in some cancers.

Animal morphogenesis is largely driven by concerted changes in the shape of individual cells. However, how cell shape changes are regulated and coordinated in developing animals is not well understood. Here we show that the two perpendicular signaling gradients of the morphogens Dpp, a TGF-β homologue, and Wingless, a Wnt family member, maintain tissue homoeostasis and control cell shape changes in the developing Drosophila wing. Clones of cells lacking Dpp or Wingless signaling invaginate apically, shorten apico-basally and subsequently extrude basally without disruption of the epithelium. During early larval development, the onset of Dpp and Wingless signaling correlates with the cuboidal-to-columnar cell shape transition of wing disc cells. Gradients in apical-basal length of columnar cells correlate during late larval development with the gradients of Dpp and Wingless signaling activities. Cells receiving high levels of Dpp and Wingless signaling are most elongated and apically constricted. Low levels of Dpp and Wingless signaling correlate with a shorter and apically wider cell morphology. Dpp and Wingless signaling is cell-autonomously required for maintaining the elongated columnar cell shape of late larval wing disc cells. Overactivation of these pathways results in precocious cell elongation during early larval development. These morphogenetic responses to Dpp and Wingless require the transcription factor complexes Mad and Tcf/β-catenin, respectively, indicating that they are mediated by changes in gene expression. The morphogenetic function of Wingless is in part mediated by one of its target genes, the transcription factor Vestigial. Wingless signaling promotes an enrichment of E-cadherin at the adherens junctions, and we show that E-cadherin is required to maintain apical-basal cell length. Dpp signaling controls the subcellular distribution of the activities of the small GTPase Rho1 and the regulatory light chain of non-muscle myosin II (MRLC). Alteration of Rho1 or MRLC activity has a profound effect on apical-basal cell length. Finally, we demonstrate that a decrease in Rho1 or MRLC activity rescues the shortening of cells with compromised Dpp signaling. Our results identify cell-autonomous roles for Dpp and Wingless signaling in promoting and maintaining the elongated columnar shape of wing disc cells. Furthermore, they suggest that Dpp and Wingless signaling control cell shape by regulating the actin-MyosinII/E-cadherin network. / Morphogenese in Tieren wird in hohem Maße von konzertierten Zellformveränderungen einzelner Zellen bewirkt. Es ist jedoch noch nicht hinreichend verstanden, wie Zellformveränderungen in sich entwickelnden Tieren reguliert und koordiniert werden. Hier zeigen wir, dass die zwei zueinander senkrecht stehenden Signalgradienten der Morphogene Dpp, eines TGF-β Homologs, und Wingless, eines Mitglieds der Wnt Familie, im sich entwickelnden Drosophila-Flügel Gewebe-Homöostase aufrechterhalten und Zellformveränderungen kontrollieren. Klone von Zellen, denen Dpp oder Wingless Signalaktivität fehlt, invaginieren von ihrer apikalen Seite her, verkürzen sich in apiko-basaler Richtung und extruieren im Folgenden auf der basalen Seite des Epithels, ohne es zu zerstören. Während der frühen Larvalentwicklung korreliert das Anschalten der Dpp und Wingless Signale mit der Zellformveränderung der Flügelscheibenzellen von kuboidal zu kolumnar. Gradienten in der apiko-basalen Länge von kolumnaren Zellen korrelieren während der späten Larvalentwicklung mit den Gradienten der Dpp und Wingless Signalaktivitäten. Zellen, die hohe Werte an Dpp und Wingless Signalen empfangen, sind am meisten elongiert und apikal konstringiert. Niedrige Werte von Dpp und Wingless Signalen korrelieren mit kürzerer und apikal weiterer Zellmorphologie. Dpp und Wingless Signale werden zellautonom gebraucht für die Aufrechterhaltung der elongierten Zellform von späten larvalen Flügelscheibenzellen. Die Überaktivierung dieser Signalwege führt zu vorzeitiger Zellverlängerung während der frühen Larvalentwicklung. Diese morphogenetischen Antworten auf Dpp und Wingless benötigen die Transkriptionsfaktor-Komplexe Mad beziehungsweise Tcf/β-catenin, was darauf hindeutet, dass sie durch Änderungen in der Genexpression vermittelt werden. Die morphogenetische Funktion von Wingless wird teilweise durch eines seiner Zielgene, Vestigial, vermittelt. Wingless Signale fördern die Anreicherung von E-cadherin an den Adherensverbindungen. Wir zeigen hier, dass E-cadherin gebraucht wird, um apiko-basale Zelllänge aufrechtzuerhalten. Dpp Signale kontrollieren die subzelluläre Verteilung der Aktivitäten der kleinen GTPase Rho1 und der regulatorischen leichten Kette von nicht-muskulärem Myosin II (MRLC). Eine Änderung in der Rho1 oder MRLC Aktivität hat weitreichende Auswirkungen auf die apiko-basale Zelllänge. Schließlich zeigen wir noch, dass eine Verringerung der Rho1 oder MRLC Aktivitäten die Zellverkürzung von Dpp-Signal kompromittierten Zellen rettet. Unsere Resultate identifizieren zellautonome Rollen für Dpp und Wingless Signale in der Förderung und Aufrechterhaltung der elongierten kolumnaren Zellform von Flügelimaginalscheibenzellen. Darüber hinaus suggerieren sie, dass Dpp und Wingless Signale die Zellform durch die Regulierung des Aktin-MyosinII/E-cadherin-Netzwerks kontrollieren.

Thesis (PhD ) -- Stellenbosch University, 2011. / Bibliography / ENGLISH ABSTRACT: The regulation of cardiac contractility is dependent on cooperative interaction between the thick and thin filaments, as well as their accessory proteins, within the cardiac sarcomere. Alteration in cardiac contractility due to a defective sarcomere typically results in cardiomyopathies, such as hypertrophic cardiomyopathy (HCM). One of the sarcomeric genes frequently mutated and which accounts for the second most common form of HCM encodes cardiac myosin binding protein C (cMyBPC), a thick filament accessory protein whose physiological function is poorly understood. However, studies have implicated cMyBPC in thick filament structure and function as well as in the regulation of contractility. The N-terminal region of cMyBPC houses the cMyBPC-motif, which contains three phosphorylation sites, between domains C1 and C2. The hierarchical phosphorylation of this motif, by first calcium/calmodulin kinase II (CamKII) and then by cyclic AMP-activated protein kinase (PKA), is cardinal in the role of cMyBPC in the regulation of cardiac contractility in response to ß-adrenergic stimulation. Moreover, phosphorylation of this motif is inversely correlated to cMyBPC proteolysis and has been shown to be cardioprotective. Thus, proteins that have an effect on cMyBPC function or turnover may also influence filament structure and hence affect contractility, which, in turn, affects the structure of the cardiac muscle. One such protein is the Copper metabolism MURR1-domain containing protein 4 (COMMD4), which was previously identified as a novel interactor of cMyBPC during a yeast two-hybrid (Y2H) library screen in our laboratory. COMMD4 binds specifically to the cMyBPC motif in a phosphorylation-dependent manner. The exact function of COMMD4 is unknown; however, it is a member of the COMM family of proteins that has been linked to copper metabolism as well as to the ubiquitin-proteasome pathway (UPS). Intriguingly, recent studies have shown that the UPS plays a role in cMyBPC-derived HCM, while dietary copper depletion is also known to cause cardiac hypertrophy. Based on these findings, COMMD4 was considered an interesting candidate regulator of sarcomeric function and contractility, and by extension, a candidate modifier of cardiac hypertrophy. Thus, the aim of the present study was two-fold. Firstly, COMMD4 was used as bait in a Y2H library screen to determine its distal ligands, with a view to further elucidate its function, particularly in the context of MyBPC functioning, and identified interactors were subjected to further in vitro and in vivo verification studies. Also, the phosphorylation-dependent nature of the interaction between COMMD4 and cMyBPC was further investigated using a domain/phosphorylation assay. Secondly, COMMD4 and its Y2H-identified putative interactors were assessed as possible modifiers of hypertrophy in a family-based association study, using three cohorts of South African HCM-families in which one of three founder mutations segregate. Six putative interactors, viz. cardiac actin (ACTC1), Down syndrome critical region 3 (DSCR3), enolase 1 (ENO1), F-box and leucine rich repeat protein 10 (FBXL10), legumain (LGMN) and sorting nexin3 (SNX3) were identified and confirmed as COMMD4 interactors using Y2H analyses, followed by in vitro and in vivo co-immunoprecipitation and 3D co-localisation assays. Moreover, as some COMMD protein family members and the newly-identified interactors of COMMD4 have previously been linked to the UPS, the functional effect of siRNA-mediated knockdown of COMMD4 on cMyBPC turnover was also investigated. Data revealed accumulation of cMyBPC in the endosomes upon COMMD4 knockdown, suggesting a functional role for COMMD4 in the turnover of cMyBPC. In addition, association analysis revealed strong evidence of association between various single nucleotide polymorphisms (SNPs) in SNX3 and a number of hypertrophy traits, thus suggesting a role for SNX3 as a candidate modifier of hypertrophy in HCM. No evidence of association was observed for any of the genes encoding the other COMMD4 interactors implicated in protein turnover. The present study demonstrates that COMMD4, a little understood member of the COMM family of proteins, binds to the cMyBPC motif of cMyBPC in a phosphorylation-dependent manner. Furthermore, based on the functions of its protein interactions, we hypothesise that COMMD4 plays a role in protein trafficking and turnover. More specifically, COMMD4 seems to help to facilitate formation of protein complexes with the Skp1-Cul1-Fbxl (SCF) E3 ubiquitin ligase and probably helps to stabilise the target substrate for subsequent ubiquitin-conjugation. As COMMD4 seems to affect the protein turnover of cMyBPC and possibly other sarcomeric proteins, such as actin, these results establish a novel association between the sarcomere, HCM and the UPS. In addition, identification of SNX3 as a hypertrophy modifier will allow for the improved understanding of HCM patho-aetiology. SNX3 thus adds to the growing body of sarcomeric modifier genes, which, eventually, may improve risk profiling in HCM. Furthermore, as genetic modifiers appear sufficient to completely prevent disease expression in some HCM carriers, the identification of SNX3 may point to the protein turnover pathway as a potential new target for intervention. / AFRIKAANSE OPSOMMING: Die regulering van kardiale kontraktiliteit is afhanklik van die koöperatiewe interaksie tussen die dik en dun filamente, asook hul geassosieerde proteïene, in die kardiale sarkomeer. Veranderinge in kardiale kontraktiliteit as gevolg van 'n defektiewe sarkomeer lei tot kardiomiopatieë soos hipertrofiese kardiomiopatie (HKM). Een van die sarkomeriese gene wat dikwels gemuteer is en wat verantwoordelik is vir die tweede algmeenste vorm van HKM,is dié van kardiale miosien-bindingsproteïen C (cMyBPC),'n proteïen geassosieer met die dik filament waarvan die fisiologiese funksie nog nie goed bekend is nie. Studies betrek cMyBPC in dik filament struktuur en funksie asook in die regulering van kontraktiliteit. Die N-terminale gebied van cMyBPC huisves die cMyBPC-motief, wat drie fosforilerings-setels tussen domeine C1 en C2 bevat. Die hiërargiese fosforilering van hierdie motief, eerstens deur kalsium/kalmodulien-gereguleerde kinase II (CamKII), gevolg deur siklies AMP-geaktiveerde proteïen kinase (PKA), is kardinaal in die rol van cMyBPC in die regulering van kardiale kontraktiliteit in reaksie op ß-adrenergiese stimulasie. Verder, fosforilering van hierdie motief is omgekeerd gekorreleer aan cMyBPC proteolise en is ook bewys om kardiobeskermend te wees. Dus, proteïene wat 'n uitwerking het op die funksie van cMyBPC mag ook filament struktuur en kontraktiliteit beïnvloed, wat op hul beurt die struktuur van die kardiale spier affekteer. Die koper metabolisme MURR1-domein bevattende protein 4 (COMMD4), was voorheen geïdentifiseer as 'n nuwe bindingsgenoot van cMyBPC tydens gis twee-hibried (G2H) analise in ons laboratorium. COMMD4 bind spesifiek aan die cMyBPC motief in 'n fosforilasie afhanklike wyse. Die presiese funksie van COMMD4 is onbekend; maar dit is 'n lid van die COMM domein familie van proteine wat geassosieerd is met koper metabolisme sowel as die “ubiquitin” proteosoom pad (UPP). Interesant genoeg, onlangse studies het bewys dat die UPP 'n rol speel in cMyBPC-afgeleide HKM, terwyl koper uitputting in die dieet ook bekend is om kardiale hipertrofie te veroorsaak. Gebaseer op hierdie bevindinge was COMMD4 oorweeg as 'n interessante kandidaat reguleerder van sarkomeries funksie en kontraktiliteit, asook 'n kandidaat modifiseerder van kardiale hipertrofie. Dus, die doel van die huidige studie was tweeledig. Eerstens, was COMMD4 as aas gebruik in 'n G2H biblioteek sifting om sy distale ligande te bepaal, met die oog om verdere lig te werp op sy funksie, veral in die konteks van MyBPC funksionering, en geïdentifiseerde bindingsgenote was onderwerp aan verdere 'in vitro’ en 'in vivo’ verifikasie studies. Daarbenewens was die fosforilering-afhanklike aard van die interaksie tussen COMMD4 en cMyBPC verder ondersoek met behulp van 'n domein/fosforilasie toets. Tweedens, COMMD4 en sy G2H-geïdentifiseerde vermeende bindingsgenote was geassesseer as moontlik modifiseerders van hipertrofie in 'n familie-gebaseerde assosiasie studie, met behulp van drie kohorte van Suid-Afrikaanse HKM-families waarin een van die drie stigter mutasies segregeer. Ses vermeende interaktors, nl. kardiale aktien (ACTC1), Down-sindroom kritiese streek 3 (DSCR3), enolase 1 (ENO1), F-boks en leusien ryke herhalings proteïen 10 (FBXL10), legumain (LGMN) en sorteer nexin3 (SNX3) is geïdentifiseer en bevestig as COMMD4 bindingsgenote deur G2H analises, gevolg deur in vitro en in vivo ko-immunopresipitasie en 3D ko-lokalisasie toetse. Die funksionele effek van siRNA-bemiddelde uitklop van COMMD4 op cMyBPC omset was ook ondersoek omdat 'n paar COMMD proteïen familielede, asook die nuut-geïdentifiseerde bindingsgenote van COMMD4, geassosieerd is met die UPP. Data toon ophoping van cMyBPC in die endosome by COMMD4 uitklop, wat dus aandui op 'n funksionele rol vir COMMD4 in die omset van cMyBPC. Daarbenewens, toon assosiasie analise sterk bewyse van assosiasie tussen die verskillende enkele nukleotied polimorfismes (SNPs) in SNX3 en 'n aantal hipertrofiese kenmerke,wat aandui op 'n rol vir SNX3 as 'n kandidaat modifiseerder van hipertrofie in HKM. Geen bewyse van assosiasie was waargeneem vir enige van die gene wat kodeer vir die ander COMMD4 bindingsgenote wat geïmpliseer word in die proteïen omset. Die huidige studie toon dat COMMD4, 'n min verstaande lid van die COMM familie van proteïene, aan die cMyBPC motief van cMyBPC in'n fosforilasie-afhanklike wyse bind. Verder, gebasseer op die funksies van die proteïen interaksies, hipotiseer ons dat COMMD4 'n rol speel in proteïen vervoer en omset. Meer spesifiek, COMMD4 blyk om die vorming van proteïene komplekse met die Skp1-Cul1-Fbxl (SCF) E3 "ubiquiti". ligase te fasiliteer en help waarskynlik om die teiken-substraat vir die daaropvolgende ubiquitin-konjugasie te stabiliseer. Omdat dit lyk asof COMMD4 die proteïen-omset van cMyBPC en moontlik ander sarkomeriese proteïene, soos aktien, ook beïnvloed, vestig die resultate dus 'n nuwe assosiasie tussen die sarkomeer, HKM en die UPP. Daarbenewens sal die identifisering van SNX3 as 'n hipertrofie modifiseerder voorsiening maak vir die verbeterde begrip van HKM pato-etiologie. SNX3 voeg dus by tot die groeiende ?getal van sarkomeriese modifiseerende gene, wat uiteindelik, die risiko-ontleding in HKM mag verbeter. Verder, omdat dit blyk dat genetiese modifiseerders voldoende is om die siekte-uitdrukking heeltemal te verhoed in sekere HKM draers, kan die identifikasie van SNX3 na die proteïen-omset roete dui as 'n potensiële nuwe teiken vir intervensie.

Skeletal muscle dysfunction in chronic obstructive pulmonary disease (COPD) is a condition in which peripheral skeletal muscle undergoes myopathic changes which impair muscle function, limit physical performance, and can lead to significant disability. While the etiology of the dysfunction is unknown, this study was conducted to test the hypothesis that chronic hypoxemia leads to alterations in oxygen transport and muscle function. A primary objective was to validate elastase-induced emphysema in rats as an animal model of skeletal muscle dysfunction in COPD.Arterial blood gases were used to determine the severity of hypoxemia and sodium dodecyl sulfate- polyacrylamide gel electrophoresis was used to determine the proportions of myosin heavy chain isoforms I, IIa, IIx, and IIb. Measures of microvascular oxygenation and blood flow in the spinotrapezius muscle allowed for determination of both convective and diffusive oxygen supply to the muscle, as well as calculation of muscle oxygen consumption at rest and during electrically stimulated three-minute muscle contractions. Muscle performance measures included peak force, force-time integral, and fatigue index. Due to a presumed rat respiratory virus, which likely resulted in the control group being nearly as hypoxemic as the elastase-induced emphysema group, this study was not able to definitively test the hypothesis that chronic hypoxemia leads to both a diminished supply and demand of oxygen in skeletal muscle. Although many of the results of the present study were not statistically significant, they exhibited consistent trends over time and are likely of physiological significance. All measures of muscle performance were lower in the emphysema group. In addition, spinotrapezius muscle oxygen consumption and blood flow were lower in the emphysema group. The addition of supplemental oxygen during isolated, small-muscle mass exercise did increase the force-time integral by ~18% in both groups, suggesting that muscle work in these hypoxemic animals may be limited by oxygen supply. Thus, the data on muscle fiber type, oxygen consumption and muscle performance suggest that elastase-induced emphysema in rats leads to a similar skeletal muscle dysfunction that is observed in humans with COPD, and indicates that it is a valid animal model of skeletal muscle dysfunction in COPD.