Structure And Function Of Unconventional Myosins and CARMIL Proteins

缺乏肌球蛋白Va（稀释）的小鼠表现出被稀释的毛色（例如，黑色小鼠变成灰色）。这种缺陷是由于黑素细胞（MCs）内黑素小体（MSs）细胞内分布的缺陷：与正常的MSs在树突状尖端的积累不同，MS的正常部位从MCs转移到周围的角质形成细胞（KCs），稀MCs中的MSs集中在MCs的中心细胞质中。对于第二个突变，稀释抑制因子（dsu）纯合子的稀释小鼠，表现出毛色的恢复（灰色恢复为黑色），但令人惊讶的是，MS在MCs中的分布没有恢复-它们仍然集中在MCs的中心细胞质中。Dsup（也被称为黑色素调节素）是一种新的、高电荷的22 kda多肽，Dsup的表达缺失挽救了稀释小鼠的毛色（PNAS, 2005）。我们现在发现dsup是MCs中唯一的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，并可能与胆固醇相互作用。FRAP分析表明dsup很少离开MS膜。重要的是，在WT、稀释和稀释/dsu小鼠的耳皮肤上观察MC细胞形状和MS在MC和KCs内的分布表明，dsu通过允许集中在稀释/dsu MCs中心的MS转移到MC细胞体周围的KCs，从而挽救了毛色而不破坏MCs内的MS分布。此外，对肌球蛋白Va呈WT型的dsu小鼠毛发和耳部皮肤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。总之，这些结果调和了稀释/dsu小鼠的动物表型和细胞表型之间的明显差异，并表明该MS膜蛋白是MS向KCs转移的负调节因子。

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.