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The function of lysosome-related organelles such as melanosomes in melanocytes, and lytic granules in cytotoxic T lymphocytes is disrupted in Griscelli syndrome and related diseases. Griscelli syndrome results from loss of function mutations in either the RAB27A (type 1 Griscelli syndrome) or MYO5A (type 2 Griscelli syndrome) genes. Melanocytes from Griscelli syndrome patients and respective murine models ashen (Rab27a mutant), dilute (myosin Va mutant), and leaden exhibit perinuclear clustering of melanosomes. Recent work suggests that Rab27a is required to recruit myosin Va to melanosomes, thereby tethering melanosomes to the peripheral actin network and promoting melanosome retention at the tips of melanocytic dendrites. Here, we characterize the function of the leaden gene product. We show that Rab27a, but not myosin Va, can be localized to melanosomes in leaden melanocytes, suggesting that the leaden gene product acts downstream of, or in parallel to, Rab27a in melanocytes to promote recruitment of myosin Va to melanosomes. We also observed reduced levels of myosin Va protein in leaden and ashen melanocytes, suggesting that myosin Va stability is influenced by the leaden and ashen gene products. In leaden cytotoxic T lymphocytes, we observed that lytic granules polarize towards the immunological synapse and kill target cells normally. However, in contrast to melanocytes, we found that neither the leaden gene product (melanophilin) nor myosin Va was detectable in cytotoxic T lymphocytes. These results suggest that Rab27a interacts with different classes of effector proteins in melanocytes and cytotoxic T lymphocytes.
Lysosome-related organelles serve a variety of specialized functions in differentiated cell types (reviewed in 1–3). Examples of lysosome-related organelles include melanosomes in melanocytes, lytic granules in cytotoxic T lymphocytes (CTLs) and dense granules in platelets. These organelles are considered to be lysosome related due to their acidic pH and the presence of lysosome markers such as lysosome-associated membrane protein (LAMP) family members. However, each also contains a unique sets of proteins important for their specialized function, e.g. tyrosinase in melanosomes and perforin in lytic granules.
Melanosomes are sites of pigment production and storage, and reside within melanocytes and retinal pigmented epithelial cells. Melanosome biogenesis is a poorly understood process that is divided into four stages according to morphological criteria [reviewed in (3)]. In mammalian skin, mature melanosomes are transported from the Golgi region to the tips of melanocyte dendrites and then transferred to adjacent keratinocytes or growing hair shafts at the hair bulb [reviewed in (4)]. This process is essential for normal pigmentation and seems to be achieved by coupling long-range bi-directional microtubule-based transport, which delivers melanosomes to the tips of dendrites, with an actin-based retention system that prevents the microtubular system from returning the melanosomes back towards the cell body (3,5,6).
CTLs are important for the destruction of tumorigenic and virally infected cells. In common with other cells of hemopoietic lineage, CTLs identify target cells and secrete soluble proteins that promote target cell lysis and apoptosis such as granzymes, and cell surface exposure of proteins, which trigger target cell apoptosis such as Fas ligand. These lytic components are stored in secretory lysosome-related granules (lytic granules) whose synthesis is triggered by target major histocompatibility complex (MHC) recognition through the T-cell receptor (TCR) [reviewed in (7)]. Conjugation of CTL with a target cell results in the formation of a highly organized three-dimensional structure known as the immunological synapse (8). Lytic granules then undergo kinesin-driven microtubule-dependent transport towards the synapse where they release their contents [reviewed in (9)].
Defects in the function of lysosome-related organelles underlie several human genetic diseases such as Chediak–Higashi syndrome (CHS), Hermansky–Pudlak syndrome (HPS) and Griscelli syndrome (GS) [reviewed in (3,10)]. Each disease affects the function of melanosomes, lytic granules, platelet dense granules and other lysosome-related organelles, to a greater or lesser extent. Recently, several of the genes mu-tated in these diseases, and respective mouse models, have been identified and this has proved insightful in terms of understanding both the molecular mechanisms underlying disease symptoms and the biology of these organelles (3, 10).
GS patients display partial albinism and immunodeficiency (11). Mutation of either of two genes causes GS: RAB27A mutations are most common and result in type 1 (typical) GS, while MYO5A (encoding myosin Va) mutations are less frequent and result in a disease involving primary neurological rather than immunological defects (type 2 GS) (12). Three naturally occurring mouse mutations are models of GS: ashen (Rab27a ash) encoding Rab27a, dilute (Myo5a d) encoding myosin Va, and leaden (Mlph ln) encoding melanophilin (13–15).
Rab27a is a member of the Rab family of small GTPases, which are important regulators of membrane transport [reviewed in (16–18)]. CTLs from GS patients and ashen mice are unable to kill target cells due to defects in the release of lytic granule contents into the immunological synapse, implicating Rab27a as a regulator of a late step in granule secretion (12,19,20). Mutations in myosin Va do not affect CTL function (12,19). Melanocytes from GS patients and ashen mice exhibit perinuclear clustering of melanosomes (15,21–23). The two other GS mouse model mutants, dilute and leaden, exhibit a similar phenotype, suggesting that the products of all three genes are involved in mediating the peripheral actin-based retention of melanosomes. This hypothesis is supported by the findings that the coat color dilution in all three mutants is suppressed, albeit to differing extents, by the dilute suppressor mutation (24) and that the dilute gene product (myosin Va) is present with Rab27a in immune complexes precipitated from melanocyte extracts (22). At present, there is no evidence to indicate whether the interaction of myosin Va and Rab27a is direct. Given the genetic evidence, one possibility is that the leaden gene product mediates the interaction of Rab27a and myosin Va.
In this work, we address the function of the leaden gene in melanocytes and CTLs. Our findings indicate that leaden may act either together with, or as an effector of Rab27a to recruit myosin Va to melanosomes while its activity is not critical to CTL function, suggesting that Rab27a operates via different effectors in different cell systems.
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We present evidence suggesting that the leaden gene product plays an important role in melanosome transport in skin melanocytes but not in CTL lytic granule secretion. Our data are consistent with the possibility that leaden acts downstream or in parallel with Rab27a to promote the recruitment of myosin Va to melanosomes, thereby tethering melanosomes to actin filaments. As Rab27a, but not leaden or myosin Va, is important for lytic granule secretion in CTLs, our data further suggest that Rab27a acts through different effector proteins in these different cell types.
We first examined the distribution of Rab27a and myosin Va in primary cultures of leaden melanocytes by immunofluorescence and immunoelectron microscopy (Figures 1 and 2). Using both methods, we observed that Rab27a, but not myosin Va, could be localized to the surfaces of melanosomes in these cells. These observations argue against the idea that the leaden gene product acts upstream of Rab27a as a targeting receptor or GDP/GTP exchange factor that would recruit and stabilize Rab27a at the cytoplasmic leaflet of melanosomes, as this model predicted that Rab27a would be unable to associate with leaden melanosomes. Also, our data do not support a function for leaden downstream of myosin Va allowing tethering of melanosomes to the peripheral actin cytoskeleton, as this model predicted that myosin Va would associate with leaden melanosomes. Instead, the present data suggest that leaden gene product is likely to act downstream of (or in parallel to) Rab27a, promoting the recruitment of myosin Va with melanosomes. Although we have previously reported the presence of myosin Va in complexes precipitated from melanocytes using anti-Rab27a antibodies (22), suggesting that the two proteins interact, there is presently no evidence to indicate whether this interaction is direct. The findings presented here suggest that the interaction may be indirect, possibly requiring the leaden gene product. Alternatively, myosin Va may require simultaneous interaction with multiple components of a complex present on melanosomes which contains Rab27a and the leaden gene product. The absence of any one of these components may result in the failure of myosin Va to associate with melanosomes.
This model for the function of the leaden gene product is supported by the recent mapping of the leaden mutation to exon 2 of the Mlph locus (14). This locus encodes melanophilin, a protein whose N terminus shows sequence homology to the N terminus ‘Rab binding region’ of the Rab3a effector, Rabphilin-3 (26). The leaden mutation is thought to result in production of a truncated protein lacking 7 amino acids in the N-terminal region. Although this protein has yet to be biochemically characterized, it seems likely that melanophilin binds Rab27a and functions as a Rab27a effector. Taken together, our work and that of Jenkins and coworkers suggests a model where the leaden gene product (melanophilin) is recruited by activated Rab27a to melanosomes, and in turn promotes the recruitment of myosin Va to melanosomes. These predictions should now be tested experimentally.
Another novel observation reported here is a general reduction in the intensity of myosin Va staining in leaden and ashen melanocytes (Figures 1 and 3). The residual myosin Va staining pattern is redistributed from melanosomes to a diffuse pattern throughout the cytoplasm and/or associated with a spot in the center of the cell which contains actin and from which microtubules emanate (Figure 1 and data not shown) (22). This observation is interesting in the light of findings that myosin Va undergoes tissue-specific alternative splicing within the C-terminal tail domain. This region is the most divergent between different members of the myosin superfamily and is thought to contain targeting information to associate with diverse cellular structures (27). Splicing of myosin Va transcripts in mouse and man is predicted to generate several isoforms of the protein (28,29). One isoform is highly expressed in the brain (here referred to as brain isoform), while a larger isoform containing additional exons is expressed in various tissues including melanocytes, but not in brain (here referred to as melanocyte isoform). The melanocyte isoform tail domain localizes to melanosomes when expressed as GFP fusion protein in melanocytes (6), but the GFP-tagged tail domain of the brain isoform is observed to concentrate in a pericentriolar dot consistent with the microtubule-organizing center and also throughout the cytoplasm in melanocytes (30). This pattern of localization is similar to the pattern of myosin Va distribution we observed in leaden melanocytes (Figure 1). Thus one possibility is that the residual staining observed in leaden and ashen melanocytes might result from the persistence of only the brain isoform due to preferential down-regulation or instability of the melanocyte isoform in these cells. Consistently, we observed that the leaden and ashen mutations do not affect the level of expression of myosin Va in murine brain tissue where the melanocyte specific isoform is not expressed (Figure 3B). However, we were unable to detect expression of the brain-specific isoform of myosin Va in either melan-a or leaden melanocytes using RT-PCR (data not shown). While the mechanism underlying myosin Va down-regulation is unclear, it is interesting to note that myosin Va interacts with a RING finger containing protein named BERP (31). There is evidence that such proteins act as ubiquitin ligases allowing the specific ubiquitination and degradation of the protein with which they associate [reviewed in (32)]. Thus, one possibility is that the absence of Rab27a or melanophilin promotes the interaction of myosin Va with BERP and its degradation, or alternatively that interaction with Rab27a and melanophilin is primarily required to stabilize myosin Va on melanosomes rather than for its initial targeting to melanosomes. The suggestion that the interaction of Rabs with their effectors might stabilize them is not without precedent. For instance, rabphilin-3 levels are significantly reduced in brains of Rab3a knockout mice (33). Future studies should question whether down-regulation results from defects in synthesis or stability of the myosin Va RNA/protein.
We also studied CTL function, which is dramatically affected by mutations in the RAB27A gene in type 1 GS patients and ashen mice. Interestingly, we found that CTL function is not affected in leaden mice as we observed normal CTL killing activity and lytic granule polarization in leaden CTLs. These observations parallel reports of normal CTL function in type 2 GS patients and dilute mouse (12,19). Furthermore, our inability to detect the expression of myosin Va or melanophilin in CTLs (Figure 5), suggests that these proteins are either not expressed at all or that the small amounts expressed are not physiologically relevant for lytic granule secretion.
Together, these findings raise the possibility that Rab27a acts through different groups of effectors in different cell types. In this model, Rab27a is envisioned to act in melanosome transport in melanocytes through the recruitment of Melanophilin and myosin Va, whilst in CTLs Rab27a would function in lytic granule secretion through the recruitment of different effector proteins. In accord with this idea, Melanophilin (also called Slac2-a for synaptotagmin-like protein lacking C2 domains) belongs to a family that includes several synaptotagmin-like proteins (Slp) and granuphilins (34,35). These proteins share a common synaptotagmin-like protein homology domain (SHD) structure at their N termini, which is related to the Rab-binding region of Rabphilin-3 and could preferentially mediate the function of Rab27a in different cell types (26, 36). Conversely, we suggest that myosin Va may interact with proteins other than Rab27a to fulfill its function in the brain. This possibility is supported by the observations that Rab27a is almost undetectable in this tissue when well perfused of blood (Figure 1B bottom panel) (37) and that type 1 GS patients do not show primary neurological impairment (12). Future studies will be needed to support these hypotheses.