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mauve: Biological Overview | References

Lysosome-related organelles (LROs) are endosomal compartments carrying tissue-specific proteins, which become enlarged in Chediak-Higashi syndrome (CHS) due to mutations in LYST. This study showed that Drosophila Mauve, a counterpart of LYST, suppresses vesicle fusion events with lipid droplets (LDs) during the formation of yolk granules (YGs), the LROs of the syncytial embryo, and opposes Rab5, which promotes fusion. Mauve localizes on YGs and at spindle poles, and it co-immunoprecipitates with the LDs' component and microtubule-associated protein Minispindles/Ch-TOG. Minispindles levels are increased at the enlarged YGs and diminished around centrosomes in mauve-derived mutant embryos. This leads to decreased microtubule nucleation from centrosomes, a defect that can be rescued by dominant-negative Rab5. Together, this reveals an unanticipated link between endosomal vesicles and centrosomes. These findings establish Mauve/LYST's role in regulating LRO formation and centrosome behavior, a role that could account for the enlarged LROs and centrosome positioning defects at the immune synapse of CHS patients (Lattao, 2021).

Autosomal recessive Chediak-Higashi syndrome (CHS) results from a mutation in the lysosomal trafficking regulator (LYST) or CHS1 gene and leads to partial albinism, neurological abnormalities, and recurrent bacterial infections. CHS cells have giant lysosome-related organelles (LROs), compartments that, in addition to lysosomal proteins, contain cell-type-specific proteins. LROs include melanosomes, lytic granules, MHC class II compartments, platelet-dense granules, basophil granules, azurophil granules, and pigment granules of Drosophila. Whether the giant LROs of CHS form through the excessive fusion of LROs or by inhibition of their fission is unclear (Lattao, 2021).

The compromised immune system in CHS is associated with enlarged LROs in natural-killer (NK) cells. NK cells normally become polarized with centrosomes close to their contact site with antigen-presenting cells, the immunological synapse (IS). Despite the formation of a mature IS in CHS NK cells, centrosomes do not correctly polarize and the enlarged LROs neither converge at the centrosome nor translocate to the synapse. Such findings could reflect defective microtubule (MT) organization by the centrosomes in CHS cells, and while some groups describe CHS centrosomes to nucleate fewer MTs, others report normal MT numbers, lengths, and distributions. Thus, the consequence of mutation in LYST for centrosome and MT function is unclear (Lattao, 2021).

Drosophila's LYST counterpart is encoded by mauve (mv) (CG42863). mv mutants show a characteristic eye color due to larger pigment granules, defective cellular immunity through large phagosomes, and enlarged starvation-induced autophagosomes, indicating several types of LRO are affected. The embryo's LROs are the yolk granules (YGs), which provide nutrition and energy during early development. YGs are produced and stored in the egg chamber when the yolk proteins (YPs) of follicle cells are internalized by clathrin-mediated endocytosis and trafficked through the endocytic pathway of the growing oocyte. YGs are present at the periphery of the egg until the early nuclear division cycles of the syncytial embryo, when they translocate to the interior as nuclei migrate to the embryo's cortex in nuclear division cycles 8 and 9. Nurse cells of the egg chamber also supply eggs with endoplasmic-reticulum-derived lipid droplets (LDs), which store maternally provided proteins and neutral lipids for energy and membrane biosynthesis (Lattao, 2021).

This study reveals Mauve's role in regulating LRO/YGs and MT nucleation from centrosomes through the maternal effect lethal (MEL) phenotypes of two new mutant alleles of mauve, mvrosario (mvros) and mv3. Embryos derived from mutant mv females have enlarged YGs that fuse with LDs, and this can be reverted by reducing Rab5 activity. mv-derived embryos also show compromised MT nucleation leading to defects in the embryo's mitotic cycles and cytoskeletal organization. Moreover, a requirement for Mauve in regulating MTs through the TACC/Msps pathway suggests a role for endosomal trafficking in the recruitment or maintenance of pericentriolar material (PCM) components at centrosomes (Lattao, 2021).

Previous studies of Drosophila mv mutants suggested a role for Mauve in suppressing the homotypic fusion of LROs (Rahman, 2012). This study has extended those observations by showing that Mauve also regulates heterotypic fusion between LROs and LDs and by showing that Mauve interacts with molecules that regulate the behavior of interphase and mitotic MTs. This study also shows that dominant-negative Rab5 not only rescues the LRO enlargement defect in mv-derived embryos but also ameliorates recruitment of Msps and PCM at centrosomes. The participation of LDs in LRO fusion that this study now describes could have been previously overlooked because of the lower numbers of LDs in other tissues compared with those in embryos or through specific differences in the mutant alleles under study (Lattao, 2021).

The finding that high levels of Mauve did not induce the formation of smaller sized vesicles together with live imaging of excessive fusion events of autofluorescent vesicles during oogenesis in mv mutant females are consistent with a role for Mauve as a negative regulator of vesicle fusion. The behavior of LDs and the incorporation of their content into the dramatically enlarged YGs of mv-derived embryos is also consistent with this model (Lattao, 2021).

Several lines of evidence support a role for Drosophila Mauve protein in regulating MT nucleation. First, this study found an enrichment of Mv-mCherry around the spindle and centrosomes during mitosis. Second, Mauve co-purifies with γ-tubulin and Msps. Third, the rosario phenotype of mauve-derived embryos is enhanced by mutations in d-tacc or msps, suggesting co-involvement of Mauve and the D-TACC:Msps complex in establishing and/or maintaining the MT-mediated organization of the syncytium that ensures dividing nuclei are at the cortex and endoreduplicating yolk nuclei in the interior. Fourth, embryos derived from mv mutant mothers have reduced amounts of both Msps and γ-tubulin at centrosomes, in accord with the diminished MT nucleating capacity of these centrosomes. Fifth, in line with the reduced amounts of MT nucleating molecules at centrosomes, the regrowth of de-polymerized MTs from centrosomes is compromised in mv-derived embryos (Lattao, 2021).

Mauve's co-purification with Msps, but not its D-TACC partner protein, is another indicator that Msps can exist independently of D-TACC. Indeed, Msps is present in several separate pools: independent of D-TACC at the centrosome; in complex with the D-TACC: Clathrin complex on the spindle; with the MT minus-end protein Patronin to assemble perinuclear non-centrosomal MTOCs (ncMTOCs); with the Augmin complex at kinetochores; and in complex with endosomal proteins such as Mauve. It is speculated that mutations affecting the constitution of Msps complexes at any one of these sites can affect another (Lattao, 2021).

The finding of defects in mitotic MT nucleation by centrosomes in mv-derived embryos suggests that there might be similar requirements at later developmental stages that may have been overlooked because flies can progress through most of the development without functional centrosomes (Lattao, 2021).

The increased NUF seen in mv-derived embryos is likely to be a secondary consequence of disruption to either or both membrane trafficking and mitosis. NUF was first described for the mutant of the nuf gene encoding an ADP ribosylation factor effector that associates with Rab11. Nuf protein is required to organize recycling endosomes in the coordinated processes of membrane trafficking and actin remodeling and embryos deficient for Rab11 also show a strong NUF phenotype. Together this suggests the possibility that NUF in mv mutants could result from the accumulation of endosomal components in the enlarged YGs, which would diminish numbers of recycling endosomes and their associated Rab11-Nuf complex. NUF can also occur as a Chk2 protein kinase-mediated response to DNA damage (DSBs), activated by DNA lesions at mitotic onset. However, this study found no evidence for DNA damage marked by the accumulation of phosphorylated γ-H2Av at DSBs. Finally, NUF also occurs in response to a wide range of primary or secondary mitotic defects. Indeed, failure of the sequestration of histone H2Av to LDs results in embryos that display mitotic defects, nuclear fallout, and reduced viability (Lattao, 2021).

Dominant-negative Rab5 suppresses enlarged YG formation and the mitotic defects of mv-derived embryos in accord with known roles of Rab5 at the early endosome and growing indications of a requirement for Rab5 in mitosis. Rab5 also mediates transient interactions between LDs and early endosomes that enable the transport of lipids between the two without resulting in their fusion. The possibility that Msps transiently localizes to LROs in wild-type embryos cannot be reuled out because LD-YG associations were observed in wild-type embryos and Msps is a component of LDs. The incorporation of Msps and LD markers into the enlarged YGs in mv-derived embryos is also rescued by a dominant-negative form of Rab5 and reciprocally, levels of Msps at centrosomes are restored. This suggests that mutation in mauve leads to mislocalization of Msps around YGs at the expense of its localization at the centrosome and so its availability for mitosis. Suppression of these mv phenotypes by dominant-negative Rab5 could therefore either reflect a passive restoration of the balance of Msps between YGs and spindle poles once YG fusion is prevented or a more active role of Rab5 in organizing the spindle poles (Lattao, 2021).

These findings add to a small but growing body of evidence for the roles of endocytic membrane trafficking in regulating centrosomal function (Naslavsky, 2020). There are no reports of a membrane-independent role of Rab5, although other groups have reported examples of trafficking proteins involved in MT nucleation in a membrane-independent manner, such as ALIX, a PCM component in human and fly cells, whose recruitment depends on Cnn/Cep215 and D-Spd2/Cep192. The late endosome marker Rab11 also appears to be a part of a dynein-dependent retrograde transport pathway bringing MT nucleating factors and spindle pole proteins to mitotic spindle poles. It is not clear whether Rab5-associated structures mature to Rab11-associated structures in mitosis as they do in interphase but it seems that the two vesicle types might have overlapping functions at centrosomes in mitosis. It will be of future interest to put these current findings into context with these earlier demonstrations of roles of Rab5- and Rab11-containing endosomes in spindle function (Lattao, 2021).

The dynamic relationship between endosomal trafficking and recruitment of MT nucleating molecules onto centrosomes may all have relevance for the role of LYST at the IS and how this is affected in CHS. Thus, it is conceivable that there may be a convergence of the two functions of the LYST protein in lymphocytes, both in regulating the size of LROs and in facilitating the correct positing of centrosomes and membraneous structures. Further studies will be required to clarify the precise roles of LYST in regulating vesicle trafficking and MT nucleation in this particular cell type (Lattao, 2021).

Although the results strongly indicate Mauve to act as a negative regulator of vesicle fusion, this study did not directly assess the fusion ability of LROs. In part, this was limited by the autofluorescent nature of YGs and LDs that restricted the extent to which fluorescently tagged proteins could be used to visualize membrane components of these bodies in dynamic studies. Future work should aim to complement these findings in cell culture and in cell-free systems to determine whether the involvement of both LROs and LDs is widespread. In a similar vein, it will be important to assess whether the roles of LYST proteins in regulating MT dynamics are conserved as implied by these findings. This would require carrying out studies of MT dynamics in other cell types, particularly in mammalian cells (Lattao, 2021).

Drosophila mauve mutants reveal a role of LYST homologs late in the maturation of phagosomes and autophagosomes

Chediak-Higashi syndrome (CHS) is a lethal disease caused by mutations that inactivate the lysosomal trafficking regulator protein (LYST). Patients suffer from diverse symptoms including oculocutaneous albinism, recurrent infections, neutropenia and progressive neurodegeneration. These defects have been traced back to over-sized lysosomes and lysosome-related organelles (LROs) in different cell types. This study explored mutants in the Drosophila mauve gene as a new model system for CHS. The mauve gene (CG42863) encodes a large BEACH domain protein of 3535 amino acids similar to LYST. This reflects a functional homology between these proteins as mauve mutants also display enlarged LROs, such as pigment granules. This Drosophila model also replicates the enhanced susceptibility to infections, and a defect is shown in the cellular immune response. Early stages of phagocytosis proceed normally in mauve mutant hemocytes but, unlike in wild type, late phagosomes fuse and generate large vacuoles containing many bacteria. Autophagy is similarly affected in mauve fat bodies as starvation-induced autophagosomes grow beyond their normal size. Together these data suggest a model in which Mauve functions to restrict homotypic fusion of different pre-lysosomal organelles and LROs (Rahman, 2012).

Mutations that interfere with the function of human LYST are the molecular cause underlying CHS. This study shows that phenotypes similar to those in CHS patients result from loss-of-functions mutations in mv, which encodes the closest homolog to LYST in the Drosophila genome. The original identification of the mv gene was based on its effect on eye color, which is changed in mv mutants due to their oversized pigment granules. This mirrors the oculocutaneous albinism of CHS patients caused by oversized and clumped melanosomes. Similarly, an important clinical symptom of CHS is the susceptibility to bacterial infections, which is also shared by mv mutants. Furthermore, in mv hemocytes an increased tubular morphology of lysosomes was observed, similar to the changes observed in beige macrophages. Together these morphological and phenotypic similarities support the notion that aspects of CHS can be modeled in mv flies. The unique set of molecular and genetic tools available in Drosophila suggests that this fly model will be useful for the analysis of the molecular mechanisms by which LYST homologs regulate membrane trafficking (Rahman, 2012).

Recurring bacterial infections are among the most frequent clinical complications observed in CHS patients, but is not well understood how the cell biological defects cause the enhanced susceptibility of patients for infections. Defects in phagocytosis have been considered as a possible cause. For example, changes in the phagocytosis of Staphylococcus aureus by leukocytes have been detected in some CHS patients, but several other studies found no loss of phagocytic activity in leukocytes from CHS patients. To gain further insight into the role of LYST homologs in phagocytosis this study used primary hemocytes cultured from Drosophila larvae. These cells have proven to be a useful, genetically tractable model system with markers available for different stages of phagocytosis (Rahman, 2012).

The current data indicate that Mauve is not required for the initial phagocytic uptake of bacteria into hemocytes, in agreement with previous work in human or mouse cells lacking LYST function. However, this study observed that phagocytosed bacteria were amassing in oversized late phagosomes of mv hemocytes. Accumulation of bacteria in phagosomes of CHS leukocytes has previously been observed and primarily attributed to intravacuolar bacterial proliferation. This study shows that even when heat-killed bacteria were used in phagocytosis assays, many more bacteria populated late phagosomes of mv compared to wild-type hemocytes. Furthermore, this difference was not reflective of an altered mode of initial uptake of the bacteria, as phagosomes positive for the early Avl and the intermediate Rab7 markers exhibit the normal distribution of bacterial content. Interestingly, enhanced homotypic fusion and the resulting formation of ‘megasomes’ is a hallmark of monocyte phagosomes containing Helicobacter pylori. This strategy is thought to contribute to the ability of these bacteria to evade the immune system and persist lifelong in human hosts. It is not known by which molecular mechanism H. pylori induces phagosome fusion and thus it is intriguing to speculate that these bacteria may inactivate LYST or an associated factor to promote the formation of megasomes (Rahman, 2012).

Similar to observations with mv, the LYST homolog LvsB has been proposed to function by preventing homotypic fusions of contractile vacuoles in Dictyostelium. Although cells mutant for lvsb displayed significantly enlarged contractile vacuoles, delivery of phagocytosed cargo to these vacuoles was not altered. Observations on phagosome maturation also parallel those on the maturation of secretory lysosomes in CHS cytotoxic T lymphocytes; early steps in the biogenesis of these organelles proceeded indistinguishably from wild-type. Only late in their maturation did secretory lysosomes fuse to form the giant LROs characteristic of CHS (Rahman, 2012).

Therefore, a straightforward explanation for the current data is a function of Mauve, and other LYST homologs, late during the maturation of different LROs to either directly or indirectly suppress their homotypic fusion (Rahman, 2012).

An equivalent function of Mauve may also explain the phenotypes observed during starvation-induced autophagy in mv larval fat bodies. Two key observations are the reduced intensity of LTR staining and the increase in the area of mCherry-Atg8-positive structures. The latter is unlikely to reflect simply an increase in expression of mCherry-Atg8, which is driven by Gal4 under control of a heterologous promoter. Furthermore, an increase in size was visible for mCherry-Atg8 and Rab7-positive structures by immunofluorescence and for autophagosomes morphologically identified by electron microcopy. Instead, these observations are straightforward to reconcile with the notion of Mauve functioning to restrain homotypic fusions of autophagosomes and the resulting increase in autophagosome size and ease of their detection in mv fat body cells (Rahman, 2012).

Other observations do not easily fit the notion of increased fusion events in starved mv fat bodies, however. For one, reduced LTR staining that typically labels acidic amphisomes and autolysosomes was observed. One explanation for such an observation would be a reduced rate of the fusion of autophagosomes with late endosomes and lysosomes that yield the acidified amphisomes and autolysosomes. Defects in these fusion events have been observed for subunits of the HOPS complex, which is necessary for the fusion of lysosomes with different organelles. However, none of the experimental systems in which its function has been probed has indicated a requirement of LYST for fusion with lysosomes. Alternatively, after their fusion with lysosomes, the significantly increased size of autophagosomes may result in a dampened or delayed acidification of the resulting autolysosomes thus resulting in a reduced trapping of LTR dye in those hybrid organelles (Rahman, 2012).

Such a change in acidification, whether due to the increased size of autophagosomes or due to another effect of Mauve function, could also explain the observation of a green-shift of mCherry-GFP-Atg8-labeled autophagosomes. This chimeric protein has been developed to measure cargo flux by following quenching of the pH-dependent GFP fluorescence as this indicator moves from autophagosomes, which have a pH similar to the cytosol, to acidified lysosomes. If mv autolysosomes fail to acidify as efficiently as in wild-type, the mCherry-GFP-Atg8 indicator is predicted to exhibit the observed green-shift. Thus, both the reduced LTR staining and green-shift of mCherry-GFP-Atg8-labeled autolysosomes are consistent with an acidification defect that may be a secondary effect of the exceptional size of mv autolysosomes (Rahman, 2012).

A direct inhibitory effect of LYST homologs on limiting membrane fusion is a compelling model to explain the recurring theme of oversized organelles that are observed in the diverse CHS models ranging from CHS patient cells and beige mice to lvsB mutant Dictyostelium and now the Drosophila mv mutant. However, the biochemical mechanism by which LYST homologs execute this function is not clear. One set of possible mechanisms has been suggested based on results of two-hybrid screens that detected interactions between LYST and several proteins involved in regulating membrane fusion events, including subunits of SNARE complexes. Whether LYST homologs engage these or other elements of fusion machineries in vivo to suppress inappropriate fusion of LROs remains to be discovered (Rahman, 2012).

The size of organelles is not only determined by the rate of membrane addition by fusions events, but also by the rate at which membranes are removed by fission events. The notion that LYST may contribute to membrane fission from lysosomes was first supported by the observation that LYST overexpression causes a reduction in the size of lysosomes. Furthermore, in lvsB mutant Dictyostelium cells, a defect in fission may also contribute to the failure of lysosomes to mature to post-lysosomes that fuse with the plasma membrane and recycle internalized cell surface proteins. Defects in fission also emerged as the major difference between beige and wild-type mouse cells when the kinetics were observed with which lysosomes restored their steady-state size after acute disturbances (Rahman, 2012).

The data do not help to distinguish between these two models for the molecular function of LYST homologs. The dynamics of the appearance of oversized late phagosomes strongly points to a role of Mauve in suppressing homotypic fusion or promoting fission late during phagosome maturation. Similarly, oversized autophagosomes and pigment granules may reflect a direct role of Mauve in suppressing inappropriate homotypic fusion during maturation of these organelles in wild-type cells. Alternatively, such phenotypes may be an indirect consequence of altered lysosome and LRO physiology. Distinguishing between these possibilities will require a better understanding of the molecular mechanism by which Mauve affects LRO size. The availability of the fly model may open new genetic and molecular approaches toward this goal (Rahman, 2012).

Search PubMed for articles about Drosophila Mauve

Lattao, R., Rangone, H., Llamazares, S. and Glover, D. M. (2021). Mauve/LYST limits fusion of lysosome-related organelles and promotes centrosomal recruitment of microtubule nucleating proteins. Dev Cell 56(7): 1000-1013. PubMed ID: 33725482

Naslavsky, N. and Caplan, S. (2020). Endocytic membrane trafficking in the control of centrosome function. Curr Opin Cell Biol 65: 150-155. PubMed ID: 32143977

Rahman, M., Haberman, A., Tracy, C., Ray, S. and Kramer, H. (2012). Drosophila mauve mutants reveal a role of LYST homologs late in the maturation of phagosomes and autophagosomes. Traffic 13(12): 1680-1692. PubMed ID: 22934826

date revised: 30 October 2021

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