Structure And Function Of Unconventional Myosins

哺乳动物的色素沉着是由含有色素的黑素小体从黑素细胞树突的尖端向周围的角质形成细胞的细胞间转移驱动的。黑素小体的尖端积累需要肌凝蛋白Va，因为黑素小体集中在肌凝蛋白Va缺失（稀释）小鼠的黑素细胞中心。这种分布缺陷导致黑素体转移效率低下和被毛颜色的稀释。同时缺乏黑色素调节素（稀释抑制基因座的产物）的稀释小鼠表现出几乎完全恢复的毛色，但令人惊讶的是，黑素体仍然集中在黑素细胞的中心。本研究显示，稀释/非稀释黑素细胞，而非稀释黑素细胞，很容易将集中在其中心的黑素小体原位转移到周围的角化细胞。利用野生型黑素细胞/角化细胞共培养的延时成像，其中两种细胞的质膜被标记为不同的颜色，然后我们定义了细胞间黑素体转移途径，该途径涉及黑素细胞脱落富含黑素体的包裹，随后被角化细胞吞噬。脱落主要发生在树突尖端，但也发生在更中心的区域，包括与角质细胞的粘附，形成包后变薄，以及明显的自我脱落。最后，我们发现，在培养的稀释/dsu黑色素细胞中，细胞中心脱落的频率是稀释黑色素细胞的6倍，与原位数据一致。总之，这些结果解释了dsu如何在不恢复细胞内黑素体分布的情况下恢复稀释小鼠的毛色，表明黑色素调节素是黑素体转移的负调节因子，并为细胞间黑素体转移的机制提供了新的见解。

Mammalian pigmentation is driven by the intercellular transfer of pigment-containing melanosomes from the tips of melanocyte dendrites to surrounding keratinocytes. Tip accumulation of melanosomes requires myosin Va, as melanosomes concentrate in the center of melanocytes from myosin Va null (dilute) mice. This distribution defect results in inefficient melanosome transfer and a dilution of coat color. Dilute mice that simultaneously lack melanoregulin, the product of the dilute suppressor locus, exhibit a near complete restoration of coat color, but surprisingly melanosomes remain concentrated in the center of their melanocytes. Here we show that dilute/dsu melanocytes, but not dilute melanocytes, readily transfer the melanosomes concentrated in their center to surrounding keratinocytes in situ. Using time lapse imaging of wild type melanocyte/keratinocyte co-cultures in which the plasma membranes of the two cells are marked with different colors, we then define an intercellular melanosome transfer pathway that involves the shedding by the melanocyte of melanosome-rich packages, which are subsequently phagocytosed by the keratinocyte. Shedding, which occurs primarily at dendritic tips, but also from more central regions, involves adhesion to the keratinocyte, thinning behind the forming package, and apparent self-abscission. Finally, we show that shedding from the cell center is six-fold more frequent in cultured dilute/dsu melanocytes than in dilute melanocytes, consistent with the in situ data. Together, these results explain how dsu restores the coat color of dilute mice without restoring intracellular melanosome distribution, indicate that melanoregulin is a negative regulator of melanosome transfer, and provide new insight into the mechanism of intercellular melanosome transfer. In mammals, pigments are made by melanocytes within a specialized organelle, the melanosome. Mature, pigment-laden melanosomes are then transferred to keratinocytes to drive the visible pigmentation of the animals hair and skin. The dilute suppressor (dsu) locus is an extragenic suppressor of the pigmentation defect exhibited by mice lacking myosin Va (i.e. dilute mice). We recently showed that melanoregulin, the product of the dsu locus, functions as a negative regulator of a shedding mechanism that drives the intercellular transfer of melanosomes from the melanocyte to the keratinocyte. Here we address melanoregulins localization within the melanocyte, as well as the molecular basis for its localization. First, we confirm and extend recently published results using exogenous, GFP-tagged melanoregulin by showing that endogenous melanoregulin also targets extensively to melanosomes. Second, using site-directed mutagenesis, metabolic labeling with H3-palmitate, and an inhibitor of palmitoylation in vivo, we show that the targeting of melanoregulin to the limiting membranes of melanosomes in melanocytes and lysosomes in CV1 cells depends critically on the palmitoylation of one or more of six closely-spaced cysteine residues located near melanoregulins N-terminus. Finally, using Fluorescence Recovery after Photobleaching (FRAP), we show that melanoregulin-GFP exhibits little if any tendency to cycle in and out of the melanosome membrane. We conclude that multiple palmitoylation serves to stably anchor melanoregulin in the melanosome membrane.