Structure And Function Of Unconventional Myosins and CARMIL Proteins

缺乏肌球蛋白Va(稀释)的小鼠表现出稀释的毛色(例如，黑色小鼠变成灰色)。这种缺陷是由于黑素细胞(MC)内黑素小体(MSS)在细胞内分布的缺陷：不是在树突尖端正常聚集MSS，而是MS的正常位置从MC转移到周围的角质形成细胞(KCs)，稀释型MC中的MSS集中在MC的中央细胞质。稀释鼠也是第二个突变纯合的稀释鼠，稀释鼠的毛色恢复(灰色恢复为黑色)，但令人惊讶的是，没有恢复MC内MS的分布-它们仍然集中在MC的中央细胞质。Dsup(也称为黑素调节蛋白)是一种新的、高度带电的22 kDa多肽，正是dsup表达的丧失挽救了稀释鼠的毛色(PNAS，2005)。我们现在发现dsup只是MC中的一种MS膜蛋白。膜靶向是由dsup的肉豆蔻酰化和多重棕榈酰化驱动的，可能是通过它与胆固醇的相互作用来驱动的。FRAP分析表明，dsup很少离开MS膜。重要的是，在WT、稀疏和稀疏/DSU小鼠的耳部皮肤中，MC细胞的形态和MS在MC和KCs中的分布的可视化显示，DSU通过允许集中在稀释型/DSDU MCs中心的MSS转移到MC细胞体周围的KCs来挽救毛色而不挽救MC内的MS分布。此外，对DSU小鼠的毛发和耳部皮肤中的色素分布的检查表明，当Dsup单独缺失时，MS向KCs转移的增加是一致的。综上所述，这些结果证实了DIMUTE/DSU小鼠动物和细胞表型的明显差异，并表明这种MS膜蛋白是MS向KCs转移的负调节因子。 DictyostelialCARMIL(DD CI)是一种116 kDa的支架蛋白，它与肌球蛋白I(MI)、帽蛋白(CP)和Arp2/3复合体在独立的位点结合。DD CI与MI、CP和Arp2/3一起定位于肌动蛋白的活性组装部位，如巨噬细胞冠和爬行细胞的前缘，DD CI缺失细胞在巨噬细胞吞噬和趋化方面存在缺陷。DD CI由一个N-末端调节域、一个中心LRR结构域和一个C-末端结构域组成，其中C-末端结构域包含与WASP的Verprolin/WH2和酸性部分相似的序列(VA结构域；Arp2/3相互作用和弱激活)，紧随其后的是一个富含Pro的结构域(P；MI SH3相互作用)，该结构域终止于CARMIL同源结构域-3(CAH3)。与哺乳动物CARMILs一样，这个80个残基的结构域与低NM亲和力的CP结合，并通过结合游离CP，从而使其对带刺末端的亲和力降低200倍(隔离活性)，并通过快速从带帽的肌动蛋白细丝末端去除CP(去封顶活性)，发挥强大的CP拮抗剂的作用。我们现在发现，全长(FL)DD CI的过度表达(OE)导致细胞丝足过剩，这一表型使人想起CP击倒后的脊椎动物细胞(Cell，2002)。与先前关于FL DD CI的抗CP活性被分子内折叠抑制的证据一致，一个N端截断的DD CI(NT DD CI)在诱导更多和更长的丝状伪足方面比FL DD CI更有效。然而，令人惊讶的是，当NT DD CI的CAH3结构域发生突变或缺失时(尽管皮质肌动蛋白斑块/凸起的诱导丢失)，这种影响仍然存在。DD CI的不同部分(LRR-VAP、VAP、VA、AP等)的OE认为，丝状孢子的诱导是由于类DD cis V/WH2序列的OE所致。目前的研究方向是确定丝状突起表型是否位于CARMIL依赖的对Arp2/3复合体活性/定位的影响的下游。

Mice that lack myosin Va (dilute) exhibit a diluted coat color (e.g. black mice become gray). This defect is due to a defect in the intracellular distribution of melanosomes (MSs) within melanocytes (MCs): instead of the normal accumulation of MSs at dendritic tips, the normal site of MS transfer from the MC to surrounding keratinocytes (KCs), MSs in dilute MCs are concentrated in the MCs central cytoplasm. Dilute mice that are also homozygous for a second mutation, dilute suppressor (dsu), exhibit a restoration of coat color (gray returns to black), but surprisingly without a restoration of MS distribution within MCs- they remain concentrated in the MCs central cytoplasm. Dsup (also known as melanoregulin) is a novel, highly charged, 22-kDa polypeptide, and it is the loss of expression of dsup that rescues the coat color of dilute mice (PNAS, 2005). We now find that dsup is exclusively a MS membrane protein in MCs. Membrane targeting is driven by myristoylation and multiple palmitoylation of dsup, and possibly by its interaction with cholesterol. FRAP analyses indicate that dsup rarely exits the MS membrane. Importantly, visualization in ear skin from WT, dilute and dilute/dsu mice of MC cell shape and MS distribution within MCs and KCs reveals that dsu rescues coat color without rescuing MS distribution within MCs by allowing the transfer of MSs concentrated in the center of dilute/dsu MCs to KCs surrounding the MC cell body. Moreover, examinations of the distribution of pigment in hair and ear skin from dsu mice that are WT for myosin Va are consistent with an augmentation of MS transfer to KCs when dsup alone is missing. Together, these results reconcile the apparent discrepancy between the animal and cellular phenotypes of dilute/dsu mice and suggest that this MS membrane protein serves as a negative regulator of MS transfer to KCs. Dictyostelium CARMIL (Dd CI) is a 116 kDa scaffold protein that binds myosin I (MI), Capping protein (CP) and the Arp2/3 complex at independent sites. Dd CI localizes along with MI, CP and Arp2/3 at sites of active actin assembly such as macropinocytic crowns and the leading edge of crawling cells, and Dd CI null cells exhibits defects in macropinocytosis and chemotaxis. Dd CI is composed of an N-terminal regulatory domain, a central LRR domain, and a C-terminal domain containing sequences resembling the verprolin/WH2 and acidic portions of WASP (VA domain; Arp2/3 interaction and weak activation) followed by a proline-rich domain (P; MI SH3 interaction) that terminates with CARMIL Homology domain-3 (CAH3). As in mammalian CARMILs, this 80-residue domain binds CP with low nM affinity and functions as a potent CP antagonist by binding free CP, thereby reducing its affinity for the barbed end 200 fold (sequestering activity), and by rapidly removing CP from the end of capped actin filaments (uncapping activity). We now find that the over expression (OE) of full length (FL) Dd CI results in cells with excess filopodia, a phenotype reminiscent of vertebrate cells following CP knock down (Cell, 2002). Consistent with previous evidence that the anti-CP activities of FL Dd CI are suppressed by intramolecular folding, an N-terminally truncated version of Dd CI (∆NT Dd CI) is much more potent than FL Dd CI at inducing more and longer filopodia. Surprisingly, however, this effect persists when the CAH3 domain of ∆NT Dd CI is either mutated or deleted (although the induction of cortical actin patches/bumps is lost). OE of various portions of Dd CI (LRR-VAP, VAP, VA, AP, etc) argues that the induction of filopodia is due to the OE of the Dd CIs V/WH2-like sequence. Current efforts are directed at determining whether the filopodial phenotype is downstream of CARMIL-dependent effects on the activity/localization of the Arp2/3 complex.