misato: Biological Overview | References
Gene name - misato
Cytological map position - 20A1-20A1
Function - chaperone
Keywords - co-factor of the Tubulin Chaperone Protein-1 (TCP-1) complex - plays an essential role in the Tubulin-folding processes required for proper assembly of spindle microtubules - depletion of Misato in the visceral muscle is a model for the pathogenic mechanism for visceral myopathy - mutations in misato inhibit kinetochore-driven microtubule growth
Symbol - mst
FlyBase ID: FBgn0020272
Genetic map position - chrX:21,388,977-21,391,533
Classification - Misato segment II tubulin-like domain
Cellular location - cytoplasmic
Mitotic spindles are primarily composed of microtubules (MTs), generated by polymerization of α- and β-Tubulin hetero-dimers. Defects in Tubulin polymerization dramatically affect spindle formation and disrupt chromosome segregation. Recently studies have described a role for the product of the conserved misato (mst) gene in regulating mitotic MT generation in flies, but the molecular function of Mst remains unknown. This study used affinity purification mass spectrometry (AP-MS) to identify interacting partners of Mst in the Drosophila embryo. Mst was shown to associate stoichiometrically with the hetero-octameric Tubulin Chaperone Protein-1 (TCP-1) complex, with the hetero-hexameric Tubulin Prefoldin complex, and with proteins having conserved roles in generating MT-competent Tubulin. RNAi-mediated in vivo depletion of any TCP-1 subunit phenocopies the effects of mutations in mst or the Prefoldin-encoding gene merry-go-round (mgr), leading to monopolar and disorganized mitotic spindles containing few MTs. Crucially, it was demonstrated that Mst, but not Mgr, is required for TCP-1 complex stability and that both the efficiency of Tubulin polymerization and Tubulin stability are drastically compromised in mst mutants. Moreover, structural bioinformatic analyses indicate that Mst resembles the three-dimensional structure of Tubulin monomers and might therefore occupy the TCP-1 complex central cavity. Collectively, these results suggest that Mst acts as a co-factor of the TCP-1 complex, playing an essential role in the tubulin-folding processes required for proper assembly of spindle MTs (Palumbo, 2015).
Previous work has demonstrated that mutations in misato (mst) lead to frequent monopolar spindles (~70%) with low microtubule (MT) density in Drosophila larval brains and that, upon MT regrowth after cold exposure, mst cells are primarily defective in kinetochore-driven MT generation (Mottier-Pavie, 2011). To address the molecular function of Mst, a GFP-tagged variant was expressed in the Drosophila syncytial blastoderm embryo, which undergoes a series of rapid, synchronous nuclear divisions. Expression of Mst-GFP fully rescued the lethality associated with the mst1 mutation and, under the control of the V32-GAL4 female germline-specific driver, Mst-GFP was expressed in embryos at similar levels to endogenous Mst. Time-lapse experiments showed that Mst-GFP is excluded from interphase nuclei. Upon nuclear envelope breakdown, Mst accumulates in the region encompassed by the mitotic spindle but is excluded from centrosomes and astral MTs. By anaphase, Mst-GFP is most intense in the region corresponding to the central spindle MTs. This dynamic localization was confirmed by analysis of fixed embryos immunostained for α-Tubulin and Mst. In contrast, the analysis of brains expressing Mst-GFP under the control of an Actin-GAL4 driver and immunolocalization studies on fixed larval brains failed to reveal Mst enrichment on the spindle. Thus, Mst is specifically associated with spindles of syncytial embryos (Palumbo, 2015).
To establish the biological process in which Mst functions, attempts were made to identify interacting partners. Syncytial embryos, which undergo 13 mitotic divisions predominantly in the absence of zygotic transcription, contain large amounts of mitotic proteins. A pipeline based on GFP-TRAP-A affinity purification and mass spectrometry (AP-MS) combined with bioinformatics-based removal of non-specific contaminants was developed that allows the robust identification of GFP-bait protein interacting partners. This procedure was undertaken in triplicate for embryos expressing Mst-GFP. Incubation of 0-3 hr clarified embryo extracts with GFP-TRAP-A consistently depleted ~95% of Mst-GFP, without affecting levels of untagged Mst, demonstrating that Mst is a monomer in the embryo. Bioinformatics-based analysis of AP precipitates identified interactors, which suggested a core functional relationship with Mst. A set of eight 'top hit' proteins had similar MS scores and peptide coverages as Mst itself (~1,300-3,000 and 70%-90%, respectively). These constitute all the subunits of the Tubulin chaperone complex, TCP-1. Also known as the Chaperonin Containing TCP-1 (CCT) or Tcp-1 Ring Complex (TRiC), the TCP-1 complex is an integral component of the Tubulin-folding pathway. In this pathway, a complex termed Prefoldin initially interacts with newly synthesized α- and β-Tubulin, delivering them to the 'donut'-shaped TCP-1 complex, which provides a suitable environment in which Tubulin can be correctly folded. A further set of conserved TCP-1 complex modulating proteins are additionally required, ensuring newly translated α- and β-Tubulin can be modified and incorporated into MTs. The same pathway controls the proper folding of Actin and γ-Tubulin (Palumbo, 2015).
All six subunits of the Prefoldin complex (see Drosophila Prefoldin 2) were also present in Mst-GFP AP precipitates. Furthermore, two of the five remaining significant hits (Viaf and PDCD-5) are TCP-1 interactors. Viaf, a member of the Phosducin family, associates with the TCP-1 complex in flies, and its closest human homolog (PhLP-3) binds TCP-1, working antagonistically to Prefoldin. Similarly, the human homolog of PDCD-5 binds to Phosducin and TCP-1β, interfering with the Tubulin-TCP-1 complex interaction (Palumbo, 2015).
The interaction between Mst and TCP-1α, the only subunit of the TCP-1 complex for which antibodies are available, was verified through reciprocal immunoprecipitation and western blotting in wild-type and Mst-GFP-expressing embryos. Thus, the interaction between the TCP-1 complex and Mst reflects a normal in vivo interaction. Moreover, quantitative analysis of the MS data confirmed that similar quantities of all eight TCP-1 complex subunits and Mst were present in AP precipitates. From this it is inferred that one molecule of Mst has the ability to interact with a single TCP-1 hetero-octameric complex. Notably, the Prefoldin subunits and the additional Tubulin-folding interactors were precipitated at ~10- to 100-fold-lower amounts than the TCP-1 subunits. This most likely reflects transient ternary complexes that are formed between these proteins and the TCP-1 complex. In summary, this study demonstrates an in vivo biochemical relationship between Mst, the TCP-1 complex, and other Tubulin-folding pathway components (Palumbo, 2015).
To investigate the functional relationship between Mst and its principal interactors, the mitotic phenotypes elicited by depletion of either Mst, the Drosophila Prefoldin 3 subunit encoded by merry-go-round (mgr), and the TCP-1 complex subunits. Available mutations in the genes encoding the TCP-1 complex subunits caused early embryonic lethality, preventing cytological analysis in larval brains. Similarly, in vivo RNAi, using UAS-RNAi lines and ubiquitous drivers, produced an early lethal phenotype. Flies were generated carrying a suitable UAS-RNAi construct and the conditional tubGAL4-tubGAL80ts driver, permitting time-restricted expression of this construct (Palumbo, 2015).
Brain preparations from larvae grown for 72 hr at 29°C, expressing UAS-RNAi constructs against the TCP-1 complex subunits, were immunostained for Tubulin and the centrosomal marker DSpd-2 and compared with mst and mgr mutant brain preparations. Consistent with previous observations, mst and mgr mutant brains displayed a metaphase arrest phenotype and frequent polyploid cells. In addition, most mst and mgr prometaphase/metaphase figures showed monopolar spindles with reduced MT density. Importantly, RNAi against Tcp-1α caused an ~80% reduction of the protein level and a mitotic phenotype indistinguishable from that elicited by mst mutations. Moreover, RNAi against each of the other seven TCP-1 subunits also resulted in a very similar phenotype; high frequencies of monopolar spindles (ranging from 39.4% to 76.7%; n = 200 per each TCP-1 subunit) were consistently observed with strongly reduced MT density (Palumbo, 2015).
To further investigate the functional relationship between Mst and the TCP-1 complex in spindle assembly, MT regrowth was analyzed after cold exposure in mitotic cells of TCP-1α-depleted brains. After cold-induced MT depolymerization, wild-type cells rapidly form new MTs, first from chromosomes and then from both chromosomes and centrosomes. The two MT populations merge within 5 min, giving rise to morphologically regular spindles. Previous work showed that mutations in mst strongly reduce chromosome-driven MT regrowth, having little effect on MT regrowth from centrosomes. Similarly, in TCP-1α-depleted brains exposed to cold and then returned at room temperature (RT), the majority of mitotic cells showed initial MT regrowth exclusively from the centrosomes; only after 10 min recovery did these cells show monopolar and bipolar spindles similar to those of untreated cells. Thus, the absence/reduction of Mst and TCP-1 subunits similarly affect spindle MT generation in larval neuroblasts, suggesting a functional link between Mst and the Tubulin-folding machinery (Palumbo, 2015).
Attempts were made to distinguish whether Mst is a substrate for the TCP-1 and Prefoldin complexes or whether it acts as a co-factor for the Tubulin-folding pathway. The amounts of Mst, Mgr, and TCP-1α were assessed in larval brain extracts depleted of each of these proteins. In extracts from RNAi larvae depleted of any TCP-1 subunit, the level of TCP-1α was always dramatically reduced. These findings indicate that all TCP-1 complex components are required for TCP-1α stability and, consistent with previous results in mammalian cells, suggest that all TCP-1 subunits are mutually required for complex stability. However, their reduction did not affect the levels of either Mgr or Mst. Remarkably, while levels of both Mst and TCP-1α were unaffected in brain extracts from mgr mutants, mutations in mst, although not affecting Mgr, caused a strong reduction in the levels of TCP-1α. These findings were corroborated by immunolocalization experiments. Similar to Mst, TCP-1α specifically accumulated in the cytoplasm of mitotic cells of wild-type brains but was almost undetectable in dividing cells of mst mutant brains (Palumbo, 2015).
To assess whether the low level of TCP-1α observed in mst mutants reflects a reduction of the entire TCP-1 complex, wild-type and mst mutant larval extracts were subjected to size exclusion chromatography, probing separated fractions for TCP-1α. The TCP-1α of mst mutants, although reduced in amount, eluted at ~500-550 kD (corresponding to the predicted size of the Drosophila TCP-1 hetero-octameric complex) with a profile identical to wild-type TCP-1α, indicating that Mst is required to stabilize the entire complex (Palumbo, 2015).
The TCP-1 complex in other experimental systems is essential for the correct folding of both Tubulin and Actin. However, while RNAi-mediated depletion of human TCP-1 complex subunits leads to a dramatic reduction in Tubulin levels, it has little effect on Actin. To determine whether Mst depletion differentially affects Tubulin and Actin in Drosophila, levels of α- and β-Tubulin, γ-Tubulin, and Actin were analyzed in wild-type and mst mutant extracts from either brains or whole larvae. Although Tubulin levels were similar in wild-type and mst mutant brains, mst larvae displayed substantially reduced levels of α- and β-Tubulin compared to wild-type larvae; this difference likely reflects variable susceptibilities of different tissues to loss of components of the Tubulin-folding pathway, as previously reported for Drosophila Mgr. In contrast, both wild-type and mst mutant brains and larvae showed very similar levels of both γ-Tubulin and Actin. Moreover, size exclusion chromatography of wild-type, mst mutant extracts, or extracts of Tcp1-α RNAi larvae failed to reveal any effect on Actin level or size distribution profile, while confirming the reduction of α-Tubulin (Palumbo, 2015).
To further investigate the specific loss and functionality of α- and β-Tubulin in mst mutant larvae, Tubulin stability and MT sedimentation assays were performed; these experiments could not be performed with isolated brains due to the difficulty in collecting enough tissue. First, by monitoring the amounts of Tubulin and Actin in extracts incubated at 27°C for varying times, it was found that, while α-Tubulin levels remained unchanged in wild-type extracts over the course of 1 hr, α-Tubulin in mst mutant extracts was rapidly lost. Next, extracts from wild-type, mst, and Tcp1-α RNAi larvae were subjected to in vitro MT and Actin sedimentation assays. Actin polymerization was unaffected in mst or Tcp1-α RNAi extracts, although a consistent band-shift, possibly reflective of post-translational modification, was observed. To control for the reduced concentration of α-Tubulin in mst and Tcp1-α RNAi larval extracts, the MT sedimentation assay was performed with various diluted wild-type extracts. Even in wild-type extracts diluted 1:16, a proportion of α-Tubulin was able to pellet under polymerization conditions. In contrast, α-Tubulin in mst mutant larval extracts, similar to that present in Tcp1-α RNAi larvae, remained in the supernatant, suggesting an inability to polymerize (Palumbo, 2015).
These data demonstrate that the functions of Mst and the TCP-1 complex are integrally linked. Mst binds the complex and is required for its stability. Moreover, Mst loss causes a mutant phenotype indistinguishable from that elicited by loss of any TCP-1 subunit, and the biochemical properties of extracts depleted of either Mst or TCP1-α are identical. Together, these results indicate that Mst directly regulates TCP-1 complex structure and function. Mst was initially described as having primary structural motifs similar to those found in the Tubulin superfamily members. Therefore this study sought to determine whether Mst could be structurally related to Tubulins at the tertiary level. To do this, a three-dimensional model of Drosophila Mst was composed. Briefly, sequences were identified homologous to Mst in the α-, β-, and γ-Tubulin and FtsZ family of proteins, the available homologous structures were structurally aligned, and Mst was merged to this alignment. This final alignment was then used for comparative modeling using the Rosetta software suite (Palumbo, 2015).
The general secondary and tertiary structural elements found within both Tubulins and FtsZ map coherently onto Mst, strongly supporting the notion that Mst is a distant member of the Tubulin superfamily. However, Mst contains additional stretches of amino acids that are predicted to be structurally disordered and that possibly affect both the nucleotide binding and oligomerization activities elicited by Tubulins. Upon superimposition of the Mst model onto the model of bovine Tubulin:TCP-1 complex, Mst was found to be capable of filling the internal cavity of the TCP-1 complex. Although its additional loops exceed the cavity boundary, it is hypothesized that the flexibility of these disordered protein stretches allows additional interactions with the TCP-1 complex. Therefore, although further work will be required to determine whether Mst does indeed sit within the TCP-1 complex cavity in vivo, the modeling is consistent with a scenario in which Mst stabilizes the TCP-1 complex in the absence of a substrate, through a Tubulin-like interaction (Palumbo, 2015).
This study also highlights the existence of tissue-specific requirements for Tubulin folding and MT polymerization. Mst was shown to associate with embryonic spindles, consistent with the report that Drosophila TCP-1 subunits co-sediment with MTs from early embryos. However, Mst does not appear to be enriched in larval brain spindles. A possible explanation for this difference is that the rapid assembly of syncytial embryonic spindles requires a high local concentration of assembly-competent Tubulin. A spindle-anchored folding machinery would, however, not be necessary in cells surrounded by a plasma membrane (such as brain cells), where sufficient folded Tubulin could be provided by increasing the intracellular concentration of the folding complexes in anticipation of mitosis (Palumbo, 2015).
Finally, the in vivo MT regrowth experiments demonstrate that, in either mst mutant or Tcp-1 RNAi background, centrosome-driven MT regrowth after cold treatment is less affected than chromosome-/kinetochore-induced regrowth. The simplest explanation is that, following cold-induced depolymerization, kinetochores and centrosomes nucleate MTs with different dynamics. Cold treatment removes the vast majority of cellular MTs, probably leaving only short, cold-resistant MT seeds at centrosomes. While rewarming of wild-type cells essentially 'reboots' spindle assembly pathways, allowing both polymerization from centrosomes and ab-initio nucleation/polymerization from kinetochores, the reduced pool of assembly-competent Tubulin present in mst or Tcp-1 mutant cells would be preferentially incorporated into the existing MT stubs at the centrosomes. An alternative hypothesis may reflect that the chromosome- and centrosome-driven MT formation pathways governing Drosophila cell division are at least in part under separate genetic control (Palumbo, 2015).
In summary, this study study identifies Mst as a factor required for the stability of the TCP-1 complex, ultimately controlling the stability and polymerizing competency of α- and β-Tubulin within the fly. Mst is a conserved protein; future work will clarify whether it is a TCP-1 complex cofactor with a mitotic role in humans (Palumbo, 2015).
Genetic mechanisms for the pathogenesis of visceral myopathy (VM) have been rarely demonstrated. This study reports the visceral role of misato (mst) in Drosophila and its implications for the pathogenesis of VM. Depletion of mst using three independent RNAi lines expressed by a pan-muscular driver elicited characteristic symptoms of VM, such as abnormal dilation of intestinal tracts, reduced gut motility, feeding defects, and decreased life span. By contrast, exaggerated expression of mst reduced intestine diameters, but increased intestinal motilities along with thickened muscle fibers, demonstrating a critical role of mst in the visceral muscle. Mst expression was detected in the adult intestine with its prominent localization to actin filaments and was required for maintenance of intestinal tubulin and actomyosin structures. Consistent with the subcellular localization of Mst, the intestinal defects induced by mst depletion were dramatically rescued by exogenous expression of an actin member. Upon ageing the intestinal defects were deteriorative with marked increase of apoptotic responses in the visceral muscle. Taken together, it is proposed that the impairment of actomyosin structures induced by mst depletion in the visceral muscle as a pathogenic mechanism for VM (Min, 2017).
Animals possess a segregated layer of musculatures called visceral muscle on the surface of gastrointestinal tract with distinct morphology and functions in comparison to the other types of musculatures such as skeletal and cardiac muscles. In particular, the visceral muscle is comprised of the circular and longitudinal muscles producing peristalsis to facilitate mechanical digestion and transportation of ingested food along intestinal tract. Besides these digestion-related functions, the visceral muscle serves as a niche for intestinal stem cells to differentiate into various intestinal cells by secreting a blend of proliferative factors. Due to these highly specialized functions, abnormalities in the visceral muscle are often associated with a spectrum of intestinal diseases in humans. Particularly, degeneration of the visceral muscle along with fibrosis is the hallmark of visceral myopathy (VM) accompanying intestinal dilation and obstruction, deficient bowel movement, abdominal pain, and malnutrition. Although VM is a rare disease, pathogenic symptoms are severe and often familial. A member of actins specific to smooth muscles was previously suggested as a causative factor for VM through genetic studies and genome-wide sequencing, however, whether intervention of a specific gene in vivo is linked to VM has remained elusive (Min, 2017).
The Drosophila intestine provides an excellent model system to investigate the genetic and pathogenic mechanism underlying VM. First of all, the Drosophila intestine preserves most aspects of the vertebrate system, including visceral muscles and epithelial intestinal cells specialized for absorbing nutrients and secreting hormonal factors. Like vertebrates, the Drosophila visceral muscle consists of inner and outer layers of circular and longitudinal muscles. Extensive studies using Drosophila have been performed to reveal how visceral muscle contributes to the cellular homeostasis in the intestine including regulation of intestinal stem cells by visceral muscle-derived factors such as Wingless/Wnt and epidermal growth factor. Besides these anatomical conservations, a plenty of genetic tools are available. For example, visceral muscle-specific driver lines, and gene silencers and activators allow one to easily intervene or potentiate expression of the genes involved in the function of the visceral muscle by driving exogenous genes and RNAi in Drosophila. Furthermore, alterations in the structure of the visceral muscle by genetic manipulations can be thoroughly traced using diverse genetic reporters in vivo (Min, 2017).
Misato (mst) encodes a protein that is highly conserved among animal species and that retains a mixture of protein motifs found in tubulins and myosins. In Drosophila, mst null mutation was shown to elicit larval lethality associated with abnormal chromosomal segregation during cell division (Miklos, 1997). Mst was also shown to regulate the formation of mitotic spindles during mitosis by interacting with the TCP-1 tubulin chaperone complex (Palumbo, 2015). However, the protein encoded by mitochondrial distribution and morphology regulator (MSTO1), the orthologue of mst in human, was shown to localize to mitochondria to regulate subcellular distribution of mitochondria and their morphology (Kimura, 2007). Studies have implicated that MSTO1 interacts with some factors including caspases, transcriptional components and actin-related proteins involved in intestinal cancer and visceral myopathy. In addition, an investigation on patients with inflammatory bowel disease revealed an SNP on the locus 1q22 containing MSTO1 24 (Min, 2017).
Through a genetic screen using a collection of RNAi libraries, this study found that depletion of mst in muscle tissues caused the VM-like traits including intestinal dilation, reduced gut motility, defective food intake, and shortened life span. The data consistently supported that mst was required for visceral muscle maintenance via controlling actomyosin structures. These results led to a proposal that the intestinal abnormalities caused by mst depletion in the Drosophila visceral muscle are a pathologic model for VM (Min, 2017).
This study shows that depletion of mst in the whole muscle tissues specifically impaired intestinal functions while skeletal muscles remained unaffected. The disrupted intestinal functions involve a spectrum of VM-like traits, such as degeneration of visceral muscle, dilation of intestinal tracts, decreased gut motility, reduced food intake and shortened life span. These pathological conditions induced by mst depletion seem to specifically correspond to the myopathic chronic intestinal pseudo-obstruction (myopathic CIPO) in human. There are two primary types of CIPO, one of which is neuropathic CIPO that results from disruption of the visceral nerves controlling the muscle contraction to generate peristalsis for mechanical digestion, and the other is called myopathic CIPO that is characterized by muscular abnormalities in the circular and longitudinal layers of visceral muscles (Antonucci, 2008). The visceral myopathic conditions along with weakened peristalsis lead to intestinal obstruction without any mechanical obstructive processes that physically block the transportation of food along the gastrointestinal tract. Although direct genetic and environmental causes for this disease have been obscure, damages on the smooth muscle of the gastrointestinal tract are a potential cause of the disease and some naturally-occurring mutations on the visceral muscle-specific actin genes are considered to be possible factors for the familiar form of myopathic CIPO. In the experimental evidence from this study, the myopathic CIPO phenotypes were similarly produced upon targeted-depletion of mst in the visceral muscle and were completely rescued by genetic restoration of mst expression, indicating that mst may specifically involves in the myopathic CIPO phenotypes. Supporting this, mst was expressed in the intestine and showed specific co-localization with the visceral actin filaments. Exaggerated expression of mst produced potentiated visceral muscle layers with thickened actin-myosin structures in contrast to mst depletion that attenuated the structures. As Mst is a highly conserved protein across various animal species, this Mst-mediated regulation of the visceral muscle may be conserved in metazoans (Min, 2017).
Intriguingly, mst depletion specifically affected visceral actin filaments even though the depletion also occurred in the skeletal muscle that harbors abundant actin filament. This led to a hypothesis that there could be a type of visceral muscle-specific actins that correlate with Mst. Although actins are highly conserved and ubiquitous cytoskeleton proteins for all tissues, some actin isoforms, such as actin gamma 2 (ACTG2) and ACTA2, are reported to be specific for aortic and enteric smooth muscles. In particular, a list of heterozygous missense variants in the ACTG2 gene was identified by extensive exome sequencing on patients with familiar forms of VM. As the control group was devoid of the ACTG2 mutations, the mutations may be strongly correlated with familiar VM. Bioinformatic alignment and calculation on pathogenic proteins for intestinal diseases predicted that MSTO1 indeed shows a close relationship with ACTG2. Interestingly, MSTO1 was linked to ACTG2 through filamin A (FLNA), an actin-binding protein that links actin filaments to the other cellular structures indicating that MSTO1 might interact with visceral actin filaments via an intermediate factor. In Drosophila, there are six genes encoding actin proteins: Act5C, Act42A, Act57B, Act87E, Act88F, and Act79B. In this study, the results support that Act79B likely interacts with Mst protein in visceral actin filaments to maintain the integrity of the visceral muscle (Min, 2017).
Mitotic spindle assembly in centrosome-containing cells relies on two main microtubule (MT) nucleation pathways, one based on centrosomes and the other on chromosomes. However, the relative role of these pathways is not well defined. In Drosophila, mutants without centrosomes can form functional anastral spindles and survive to adulthood. This study shows that mutations in the Drosophila misato (mst) gene inhibit kinetochore-driven MT growth, lead to the formation of monopolar spindles and cause larval lethality. In most prophase cells of mst mutant brains, asters are well separated, but collapse with progression of mitosis, suggesting that k-fibers are essential for maintenance of aster separation and spindle bipolarity. Analysis of mst; Sas-4 double mutants showed that mitotic cells lacking both the centrosomes and the mst function form polarized MT arrays that resemble monopolar spindles. MT regrowth experiments after cold exposure revealed that in mst; Sas-4 metaphase cells MTs regrow from several sites, which eventually coalesce to form a single polarized MT array. By contrast, in Sas-4 single mutants, chromosome-driven MT regrowth mostly produced robust bipolar spindles. Collectively, these results indicate that kinetochore-driven MT formation is an essential process for proper spindle assembly in Drosophila somatic cells (Mottier-Pavie, 2011).
This study has shown that mst function is required for chromosome-associated MT regrowth after cold exposure. Abundant evidence indicates that chromatin and kinetochores have the ability to promote MT nucleation and stabilization. Studies in Xenopus have shown that DNA injected into eggs promotes MT nucleation leading to the formation of spindle-like structures. Spindle-like structures were also observed around DNA-coated beads incubated in Xenopus egg extracts. These findings suggested that the process of spindle formation does not require the centromere-kinetochore function. However, several studies have demonstrated that in somatic cells recovering from MT poisons or cold exposure, MTs regrow from the kinetochores and not from chromosome arms. Consistent with these results, RCC1 and other factors regulating the Ran GTP-GDP cycle are enriched at mammalian kinetochores, and RanGTP accumulates at the kinetochore of cells recovering from MT depolymerization. Most importantly, there is evidence that kinetochores can drive MT growth even under physiological conditions. Studies in mammalian cells have shown that in monopolar spindles the kinetochores that face away from the centrosome can drive formation of k-fibers. Similarly, in Drosophila S2 cells, chromosomes that are distant from the astral MTs develop k-fibers from the kinetochore that does not face the centrosome. Thus, in both Drosophila and mammalian cells, chromosome-induced MT growth occurs primarily at the centromere-kinetochore region (Mottier-Pavie, 2011).
The pattern of MT regrowth observed in these experiments on wild-type neuroblasts is very similar to the pattern of kinetochore-driven formation of k-fibers observed in Drosophila S2 cells either untreated or exposed to MT-depolymerizing agents (see Bucciarelli, 2009). To explain the mechanism of kinetochore-driven k-fiber formation it has been proposed that kinetochores capture the plus ends of MTs nucleated near the centromere; these MTs continue to polymerize at the kinetochore, forming MT bundles with the minus ends pointing away from the chromosomes. Interactions between these MT bundles and the astral MTs lead to the formation of mature k-fibers that connect the chromosomes with the spindle poles. The current results are consistent with this model, and strongly suggest that mst mutants are specifically defective in kinetochore-driven but not centrosome-driven MT growth. MTs emanating from mst centrosomes eventually reach the kinetochores and form sparse k-fibers, which are much thinner than those of wild-type cells because they do not incorporate the MTs generated by the kinetochores (Mottier-Pavie, 2011).
The role of mst in kinetochore-induced MT formation is currently unknown. Studies in human cells have suggested that an Mst homologue is localized at the outer mitochondrial membrane and regulates mitochondrial distribution and morphology. However, it is unlikely that mst regulates MT growth by affecting mitochondrial functions, because this study found that Drosophila Mst is not associated with mitochondria. It was also found that Mst does not associate with MTs in pull-down assays, which is consistent with the finding that anti-Mst antibodies do not decorate the spindle MTs. In addition, preliminary co-immunoprecipitation and mass spectrometry experiments did not reveal reliable Mst interactors. Thus, the data do not provide indications on whether mst mutants are defective in chromatin-induced MT nucleation near the centromere or in the subsequent kinetochore-driven formation of k-fibers. It is also possible that the mst function is not restricted to the formation of k-fibers and that mst has functions that are general to MT behavior, such as MT dynamics or bundling. However, even if mst had a more general role in MT behavior, kinetochore-driven MTs are clearly more susceptible to the loss of mst function than are other MTs. It was found that Mst is primarily expressed during mitosis, and accumulates in dividing cells from prophase through telophase. This finding leads to a speculation that Mst might be involved in the upregulation of MT dynamics that characterizes the interphase to M-phase transition. However, the molecular mechanisms underlying the Mst-dependent MT behavior and the specific role of Mst in kinetochore-driven k-fiber formation are currently unclear and remain a matter for future studies (Mottier-Pavie, 2011).
An incomplete occupancy of the kinetochore plate by k-MTs is probably responsible for spindle assembly checkpoint SAC protein recruitment at kinetochores and SAC-mediated metaphase arrest in mst mutants. When the SAC is abrogated by a mutation in the rod gene, a fraction of the metaphases of mst mutant brains manages to undergo anaphase. However, most mutant anaphases seen in mst; rod double mutants exhibit defects in chromosome segregation that are more severe than those observed in rod single mutants, suggesting that the thin k-fibers of mst mutant cells have a reduced ability to mediate chromosome segregation. Interestingly, mst; rod double mutants displayed a lower frequency of polyploid cells than mst single mutants. A possible interpretation for this finding is that loss of rod function in an mst mutant background releases bipolar spindles from SAC-induced metaphase arrest, allowing chromosome segregation and preventing polyploid cell formation. It can be also envisaged that in mst; rod double mutants, a fraction of the mitotic cells undergoes anaphase before aster collapse, further reducing polyploid cell formation (Mottier-Pavie, 2011).
In mst mutant brains, prophase cells display normal aster separation, whereas most prometaphase-metaphase figures are monopolar. This suggests that k-fibers are required to maintain aster separation after nuclear envelope breakdown (NEB). Several studies indicate that k-fibers have a role in aster separation. However, the effects of k-fiber disruption appear to be cell-type specific. The Aurora A activator TPX2 is required for both chromosome-dependent MT formation and the assembly of a bipolar spindle. However, there is a controversy on the spindle abnormalities caused by depletion of TPX2 in human cells. A study showed that loss of TPX2 causes centrosome fragmentation and multipolar spindles in HeLa cells. In other studies on HeLa cells, TPX2 depletion resulted in two prominent asters that did not interact to form a bipolar spindle. More recent work has shown that RNAi against TPX2 in U2OS cells leads to aster collapse and monopolar spindles. In Caenorhabditis elegans embryos depleted of the TPX2 ortholog TPXL-1, asters also collapsed, but gave rise to short bipolar spindles. Another factor required for both k-fiber formation and centrosome separation is the human kinetochore protein Mcm21R/CENP-O; recent work on this protein has led to the conclusion that k-fibers use the poleward MT flux to generate forces that push centrosomes apart. Analysis of Drosophila mitosis has provided additional evidence for a role of k-fibers in centrosome separation. The conserved Drosophila Orbit/Mast protein (CLASP in vertebrates) is required for tubulin dimer addition to the MT plus ends of fluxing k-fibers and for the maintenance of kinetochore and k-fiber connection (Maiato, 2002; Maiato, 2005). Mutations in orbit/mast and RNAi-mediated knockdown of these genes cause aster collapse and frequent monopolar spindles in embryonic and S2 tissue culture cells, respectively. Finally, it has been observed that RNAi-mediated depletion of the augmin complex in S2 cells leads to a complete suppression of k-fiber regrowth after cold exposure (Bucciarelli, 2009) and to the formation of monopolar spindles. Collectively, these results indicate that in many systems, the fluxing MTs of k-fibers exert forces required to maintain proper spindle architecture. It is proposed that the thin k-fibers of mst mutant spindles are unable to generate sufficient force to keep the centrosomes apart and avoid aster collapse (Mottier-Pavie, 2011).
mst is the first Drosophila gene so far identified that appears to be specifically required for kinetochore-driven MT formation during mitosis of a living organism. The orbit/mast gene and the augmin-coding genes (wac and msd1) are not specifically involved in this process. Orbit/Mast is a microtubule-associated protein that is particularly enriched at the spindle poles and the central spindle midzone, and it is required for both spindle assembly and cytokinesis. Studies on two augmin subunits, Wac and Msd1, have shown that they are not essential for mitotic spindle formation and fly viability, but are only required for meiotic spindle organization in females. Thus, the analysis of mst mutants allow defining of the role of kinetochore-driven MT formation in living flies. Previous studies have shown that lack of centrosomal MTs results in anastral, but otherwise functional spindles, and there is evidence that flies without centrosomes can develop to adulthood. By contrast, the current results indicate that kinetochore-driven MT formation is an essential process for Drosophila mitotic division and development (Mottier-Pavie, 2011).
The specific involvement of Mst in kinetochore-driven MT formation gave led to an opportunity to ask whether Drosophila brain cells can form a spindle in the absence of both centrosomal and kinetochore-driven MTs. It was found that most mst; Sas-4 cells displayed acentrosomal MT arrays (AMTAs) that resembled monopolar spindles. MT regrowth assays showed that in mst; Sas-4 metaphases, MTs grow from multiple foci, which eventually aggregate to form a monopolar MT array that is indistinguishable from an AMTA observed in non-chilled cells. In cells returned to 25°C for 2 minutes, robust MT regrowth foci were observed that were well separated from the chromosomes. These foci are likely to contain MTs nucleated independently of the chromosomes, perhaps by MT nucleation sites associated with membrane and/or Golgi. The same cells also contained foci that appeared to lie on or near the chromosomes. However, the finding that mst single mutants are severely defective in chromosome-driven MT regrowth suggests that most, if not all, of the foci that appear to lie over the chromosomes (because of the squashing procedure used to obtain brain preparations) do in fact contain MTs that were nucleated independently of the chromatin (Mottier-Pavie, 2011).
It is thus proposed that in mst; Sas-4 mutants all MT growth foci contain MTs that were nucleated independently of the chromosomes; these foci would associate with the chromosomes through a RanGTP-dependent mechanism that attracts growing MTs towards the chromatin. Studies using an in vitro system containing Xenopus egg extracts, chromatin-coated beads and purified human centrosomes have shown that the MTs nucleated by the centrosomes grow towards the chromatin. This leads to a concomitant movement of the asters, which stops when the astral MTs are stabilized by the high RanGTP concentration around the chromatin. A Ran-GTP-mediated attraction of astral MTs has also been described in mammalian cells; mammalian chromosomes with defective kinetochores, but capable of generating a normal RanGTP gradient are unable to form k-fibers but retain the ability to attract astral MTs. Thus it is suggested that in an mst mutant background, kinetochore-driven k-fiber formation is inhibited, whereas chromosomes retain the ability to form a RanGTP gradient that attracts the MTs nucleated at either the centrosomes or other cellular sites. In the absence of centrosomes, the MTs emanating from several chromosome-independent growth foci would be attracted by the chromatin, ultimately leading to chromosome-associated AMTAs (Mottier-Pavie, 2011).
The size of acentrosomal MT arrays (AMTAs) would suggest that MTs nucleated at non-canonical sites provide a substantial contribution to mitotic spindle assembly. However, it should be considered that the relatively large size of AMTAs could be the consequence of an increase in tubulin dimer availability as a result of the inhibition of MT polymerization at both the centrosomes and chromosomes. Consistent with this view, in both mst and Sas-4 single mutants, chromosome-independent MT regrowth foci were smaller and less frequent than in the double mutants. In addition, previous studies have shown that in both mammalian and Drosophila cells, inhibition of chromosome-dependent MT formation results in overgrowth of aster. Collectively, these results indicate that centrosomes and kinetochores have dominant roles in mitotic MT nucleation over non-canonical nucleation sites (Mottier-Pavie, 2011).
Previous studies have shown that Drosophila cells depleted of factors required for proper spindle assembly can form short bipolar spindles. Even if many of the AMTAs observed in mst; Sas-4 cells comprise enough MTs to form a short bipolar spindle, in both untreated cells and cells subjected to MT regrowth, bipolar MT arrays were rare. By contrast, MT regrowth in Sas-4 single mutants mostly generated bipolar spindles. Thus, in Drosophila somatic cells the assembly of both centrosomal and acentrosomal bipolar spindles requires kinetochore-driven MT formation and cannot rely on MTs nucleated in other cellular compartments (Mottier-Pavie, 2011).
Null mutations at the misato locus of Drosophila melanogaster are associated with irregular chromosomal segregation at cell division. The consequences for morphogenesis are that mutant larvae are almost devoid of imaginal disk tissue, have a reduction in brain size, and die before the late third-instar larval stage. To analyze these findings, cDNAs in and around the misato locus were isolated, the breakpoints of chromosomal deficiencies were mapped, which transcript corresponded to the misato gene was determined, the cell division defects were rescued in transgenic organisms, and the genomic DNA was sequenced. Database searches revealed that misato codes for a novel protein, the N-terminal half of which contains a mixture of peptide motifs found in alpha-, beta-, and gamma-tubulins, as well as a motif related to part of the myosin heavy chain proteins. The sequence characteristics of misato indicate either that it arose from an ancestral tubulin-like gene, different parts of which underwent convergent evolution to resemble motifs in the conventional tubulins, or that it arose by the capture of motifs from different tubulin genes. The Saccharomyces cerevisiae genome lacks a true homolog of the misato gene, and this finding highlights the emerging problem of assigning functional attributes to orphan genes that occur only in some evolutionary lineages (Miklos, 1997).
Misato of Drosophila melanogaster and Saccharomyces cerevisiae DML1 are conserved proteins having a homologous region with a part of the GTPase family that includes eukaryotic tubulin and prokaryotic FtsZ. This study characterized human Misato sharing homology with Misato of D. melanogaster and S. cerevisiae DML1. Tissue distribution of Misato exhibited ubiquitous distribution. Subcellular localization of the protein studied using anti-Misato antibody suggested that it is localized to the mitochondria. Further experiments of fractionating mitochondria revealed that Misato was localized to the outer membrane. The transfection of Misato siRNA led to growth deficiencies compared with control siRNA transfected HeLa cells, and the Misato-depleted HeLa cells showed apoptotic nuclear fragmentation resulting in cell death. After silencing of Misato, the filamentous mitochondrial network disappeared and fragmented mitochondria were observed, indicating human Misato has a role in mitochondrial fusion. To examine the effects of overexpression, COS-7 cells were transfected with cDNA encoding EGFP-Misato. Its overexpression resulted in the formation of perinuclear aggregations of mitochondria in these cells. The Misato-overexpressing cells showed low viability and had no nuclei or a small and structurally unusual ones. These results indicated that human Misato has a role(s) in mitochondrial distribution and morphology and that its unregulated expression leads to cell death (Kimura, 2007).
Search PubMed for articles about Drosophila Misato
Antonucci, A., Fronzoni, L., Cogliandro, L., Cogliandro, R. F., Caputo, C., De Giorgio, R., Pallotti, F., Barbara, G., Corinaldesi, R. and Stanghellini, V. (2008). Chronic intestinal pseudo-obstruction. World J Gastroenterol 14(19): 2953-2961. PubMed ID: 18494042
Bucciarelli, E., Pellacani, C., Naim, V., Palena, A., Gatti, M. and Somma, M. P. (2009). Drosophila Dgt6 interacts with Ndc80, Msps/XMAP215, and gamma-tubulin to promote kinetochore-driven MT formation. Curr Biol 19(21): 1839-1845. PubMed ID: 19836241
Kimura, M. and Okano, Y. (2007). Human Misato regulates mitochondrial distribution and morphology. Exp Cell Res 313(7): 1393-1404. PubMed ID: 17349998
Miklos, G. L., Yamamoto, M., Burns, R. G. and Maleszka, R. (1997). An essential cell division gene of Drosophila, absent from Saccharomyces, encodes an unusual protein with tubulin-like and myosin-like peptide motifs. Proc Natl Acad Sci U S A 94(10): 5189-5194. PubMed ID: 9144213
Min, S., Yoon, W., Cho, H. and Chung, J. (2017). Misato underlies visceral myopathy in Drosophila. Sci Rep 7(1): 17700. PubMed ID: 29255146
Mottier-Pavie, V., Cenci, G., Vernì, F., Gatti, M. and Bonaccorsi, S. (2011). Phenotypic analysis of misato function reveals roles of noncentrosomal microtubules in Drosophila spindle formation. J. Cell Sci. 124(Pt 5): 706-17. PubMed ID: 21285248
Palumbo, V., Pellacani, C., Heesom, K. J., Rogala, K. B., Deane, C. M., Mottier-Pavie, V., Gatti, M., Bonaccorsi, S. and Wakefield, J. G. (2015). Misato controls mitotic microtubule generation by stabilizing the TCP-1 tubulin chaperone complex [corrected]. Curr Biol 25(13): 1777-1783. PubMed ID: 26096973
date revised: 2 August 2018
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