MYL5 (1-173 a.a.) Human

What is the structural composition of human MYL5?

Human MYL5 is a 173 amino acid protein belonging to the myosin regulatory light chain (RLC) family of the MLC2 type. The protein contains three EF hand domains that are predicted to be important for calcium binding, similar to other myosin regulatory light chains . There are two recognized isoforms of MYL5: a 19.5 kD isoform (UniProtKB-Q02045-1) and a smaller 14.9 kD isoform (UniProtKB-Q02045-2) . Both isoforms maintain consistent protein levels throughout most of the cell cycle, with a slight decrease observed during mitotic exit .

How evolutionarily conserved is MYL5?

Phylogenetic analysis indicates that MYL5 is well conserved among vertebrates . This high degree of conservation suggests that MYL5 likely performs essential cellular functions that have been maintained through evolutionary processes. The conserved nature of MYL5 makes it a valuable subject for comparative studies across different vertebrate models to understand its fundamental biological roles.

Which specific cellular structures does MYL5 associate with during mitosis?

During mitosis, MYL5 co-localizes with spindle pole proteins and the unconventional myosin MYO10 . Immunofluorescence microscopy has confirmed that GFP-tagged MYL5 localizes to the mitotic spindle poles during prometaphase, metaphase, anaphase, and cytokinesis . The protein's localization signal overlaps with that of other spindle pole proteins such as NUMA and Pericentrin , suggesting functional integration with the spindle apparatus during cell division.

Does MYL5 protein expression fluctuate throughout the cell cycle?

Analysis of endogenous MYL5 protein levels throughout the cell cycle indicates that they remain relatively steady during G1/S and G2/M phases . A slight decrease in MYL5 levels is observed during mitotic exit, coinciding with the period when mitotic Cyclin B levels decrease . This pattern suggests that while MYL5 localization is strongly cell cycle-dependent, its expression levels are more consistent, with only subtle regulation during cell cycle progression.

What mitotic functions has MYL5 been implicated in?

Research has revealed that MYL5 plays critical roles in multiple aspects of mitosis. It is important for proper mitotic spindle assembly, chromosome congression, and accurate cell division . Experimental evidence indicates that MYL5 is necessary for ensuring the fidelity of these processes, with its depletion leading to various mitotic defects including multipolar spindles and errors in chromosome movements . These findings position MYL5 as a key factor in maintaining genomic stability during cellular division.

What are the consequences of MYL5 depletion on mitotic progression?

Depletion of MYL5 leads to several significant mitotic defects. Studies show an increase in prometaphase cells with multipolar spindles (approximately 20.75% compared to 7.5% in control conditions) . Additionally, there is an increase in anaphase cells with lagging chromosomes . Time-lapse microscopy has further revealed that MYL5 depletion results in a slowed progression through mitosis . These observations underscore the importance of MYL5 in maintaining proper spindle architecture and ensuring accurate chromosome segregation.

How does MYL5 overexpression affect cell division?

In contrast to the effects of depletion, overexpression of MYL5 leads to accelerated progression through mitosis . Cells overexpressing MYL5 exhibit a significantly faster time from nuclear envelope breakdown to anaphase onset and from anaphase onset to chromosome separation compared to control cells . This accelerated division suggests that MYL5 may be a rate-limiting factor in mitotic progression, with potential implications for cellular hyperproliferation in pathological contexts.

What is the relationship between MYL5 and MYO10?

MYL5 co-localizes with MYO10 (an unconventional myosin) at the spindle poles throughout mitosis . Beyond co-localization, in vitro binding experiments have demonstrated that MYL5 physically interacts with MYO10 . This interaction suggests that MYL5 may function as a regulatory light chain for MYO10 during mitosis, potentially modulating its activity in spindle assembly and chromosome movements. The MYL5-MYO10 functional partnership represents a novel regulatory mechanism in mitotic progression.

What structural domains are important for the MYL5-MYO10 interaction?

The IQ motifs of myosins are key sites for binding to regulatory light chains . Experimental evidence suggests that the three IQ motifs of MYO10 are likely necessary for the MYO10-MYL5 interaction . When these IQ motifs are removed from MYO10 (generating a MYO10 IQ-less variant), the interaction with MYL5 is affected. This structural dependency highlights the specificity of the interaction and provides insight into the molecular basis of MYL5's regulatory functions.

How do the MYL5 depletion phenotypes compare to MYO10 depletion phenotypes?

The mitotic defects observed upon MYL5 depletion are similar to those reported for MYO10 depletion, although they appear to be less severe . Both depletions lead to spindle assembly defects, errors in chromosome congression and segregation, and altered progression through mitosis . The similarity in phenotypes further supports the functional relationship between these two proteins and suggests they operate in the same pathway to ensure proper mitotic progression.

What functional roles has MYL5 been shown to play in cancer progression?

Experimental evidence indicates that MYL5 overexpression promotes tumor cell metastasis in a cervical cancer mouse model . The finding that MYL5 overexpression accelerates mitotic progression suggests it may contribute to increased proliferation rates in cancers where it is amplified. Additionally, MYL5 has been linked to increased rates of cell migration and metastasis, correlating with poor survival in certain cancer types . These observations suggest that MYL5 may influence cancer progression through multiple mechanisms, affecting both cell division and cell migration.

How might the mitotic functions of MYL5 contribute to cancer development?

The accelerated mitotic progression observed with MYL5 overexpression could potentially contribute to genomic instability and hyperproliferation in cancer cells . Since proper regulation of mitosis is essential for maintaining genomic integrity, dysregulation of MYL5 might lead to chromosomal abnormalities and aneuploidy, which are hallmarks of many cancers. Understanding how MYL5's mitotic functions relate to cancer development could provide insights into novel therapeutic approaches targeting mitotic regulators.

What are effective approaches for studying MYL5 localization?

Researchers have successfully employed several techniques to study MYL5 localization. The generation of LAP(GFP-TEV-S-Peptide)-Myl5 inducible stable cell lines has proven effective for visualizing MYL5 localization throughout the cell cycle . Fixed-cell immunofluorescence microscopy using anti-α-Tubulin antibodies to detect the mitotic microtubule spindle, combined with Hoechst 33342 DNA staining, allows for precise determination of MYL5 localization relative to other cellular structures . Co-staining with antibodies against spindle pole proteins such as NUMA and Pericentrin further enables confirmation of MYL5's association with specific subcellular structures .

How can MYL5 depletion experiments be optimized?

Effective MYL5 depletion can be achieved using RNA interference techniques. Researchers have successfully used siRNA-mediated knockdown to reduce MYL5 expression levels . The efficacy of depletion can be verified through immunoblotting with anti-MYL5 antibodies . For studying the effects of MYL5 depletion on mitosis, synchronizing cells in G1/S with thymidine treatment, followed by release into the cell cycle, allows for focused analysis of mitotic events . This approach enables the observation of specific mitotic defects that arise from MYL5 depletion.

What methods are useful for investigating MYL5-protein interactions?

In vitro binding experiments have been successfully used to demonstrate the interaction between MYL5 and MYO10 . These experiments typically involve co-immunoprecipitation of tagged proteins (such as HA-Myl5 and FLAG-MYO10) followed by immunoblot analysis . For studying the structural requirements of these interactions, modified constructs (such as MYO10 with IQ motifs removed) can be generated and tested in similar binding assays . These approaches allow for detailed characterization of the molecular interactions involving MYL5.

How might calcium signaling regulate MYL5 function in mitosis?

Recent research has shown that a calcium (Ca²⁺) signal localizes to the centrosome and spindle poles throughout mitosis and is important for mammalian cell division . Since MYL5 contains EF hand domains capable of binding Ca²⁺, and the activity of myosin light chains is influenced by Ca²⁺-binding , investigating whether the localized availability of Ca²⁺ at the spindle poles regulates MYL5 function represents an important avenue for future research. Understanding this potential regulatory mechanism could provide insights into the molecular control of mitotic progression.

What are the non-mitotic functions of MYL5 in normal and cancer cells?

While MYL5's mitotic functions have been characterized, its potential roles outside of mitosis remain largely unexplored. Given that myosins are involved in various cellular processes including cytoskeletal organization and cell migration, investigating MYL5's functions in these contexts could be valuable . Particularly in cancer, where MYL5 has been linked to metastasis, understanding how it may regulate cytoskeletal networks during cell migration could reveal new aspects of its contribution to cancer progression.

How might MYL5 be targeted therapeutically in cancer?

With MYL5 amplification observed in several cancer types and its overexpression linked to poor survival and metastasis, exploring its potential as a therapeutic target is warranted. Future research could investigate whether inhibiting MYL5-MYO10 interaction or modulating MYL5 expression levels could affect cancer cell proliferation and migration. Developing specific inhibitors or utilizing targeted gene therapy approaches could provide new strategies for cancer treatment, particularly for cancers with MYL5 amplification.