Myosin VI and its binding partner optineurin are involved in secretory vesicle fusion at the plasma membrane

Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB20XY, United Kingdom.
Molecular biology of the cell (Impact Factor: 4.47). 01/2011; 22(1):54-65. DOI: 10.1091/mbc.E10-06-0553
Source: PubMed


During constitutive secretion, proteins synthesized at the endoplasmic reticulum (ER) are transported to the Golgi complex for processing and then to the plasma membrane for incorporation or extracellular release. This study uses a unique live-cell constitutive secretion assay to establish roles for the molecular motor myosin VI and its binding partner optineurin in discrete stages of secretion. Small interfering RNA-based knockdown of myosin VI causes an ER-to-Golgi transport delay, suggesting an unexpected function for myosin VI in the early secretory pathway. Depletion of myosin VI or optineurin does not affect the number of vesicles leaving the trans-Golgi network (TGN), indicating that these proteins do not function in TGN vesicle formation. However, myosin VI and optineurin colocalize with secretory vesicles at the plasma membrane. Furthermore, live-cell total internal reflection fluorescence microscopy demonstrates that myosin VI or optineurin depletion reduces the total number of vesicle fusion events at the plasma membrane and increases both the proportion of incomplete fusion events and the number of docked vesicles in this region. These results suggest a novel role for myosin VI and optineurin in regulation of fusion pores formed between secretory vesicles and the plasma membrane during the final stages of secretion.

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    • "The down-regulation of myosin VI expression using small interfering RNA selectively reduces the secretion of prostate-specific antigen and vascular endothelial growth factor in the prostate cancer cell line LNCaP (149). Myosin VI together with its binding partner optineurin, regulates the final stage of constitutive exocytosis by mechanically controlling the formation of the fusion pore between the SV and the plasma membrane in HeLa cells (150). Less is known about the role of myosin VI in the nervous system (151). "
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    ABSTRACT: Dysregulation of regulated exocytosis is linked to an array of pathological conditions, including neurodegenerative disorders, asthma, and diabetes. Understanding the molecular mechanisms underpinning neuroexocytosis including the processes that allow neurosecretory vesicles to access and fuse with the plasma membrane and to recycle post-fusion, is therefore critical to the design of future therapeutic drugs that will efficiently tackle these diseases. Despite considerable efforts to determine the principles of vesicular fusion, the mechanisms controlling the approach of vesicles to the plasma membrane in order to undergo tethering, docking, priming, and fusion remain poorly understood. All these steps involve the cortical actin network, a dense mesh of actin filaments localized beneath the plasma membrane. Recent work overturned the long-held belief that the cortical actin network only plays a passive constraining role in neuroexocytosis functioning as a physical barrier that partly breaks down upon entry of Ca(2+) to allow secretory vesicles to reach the plasma membrane. A multitude of new roles for the cortical actin network in regulated exocytosis have now emerged and point to highly dynamic novel functions of key myosin molecular motors. Myosins are not only believed to help bring about dynamic changes in the actin cytoskeleton, tethering and guiding vesicles to their fusion sites, but they also regulate the size and duration of the fusion pore, thereby directly contributing to the release of neurotransmitters and hormones. Here we discuss the functions of the cortical actin network, myosins, and their effectors in controlling the processes that lead to tethering, directed transport, docking, and fusion of exocytotic vesicles in regulated exocytosis.
    Full-text · Article · Oct 2013 · Frontiers in Endocrinology
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    • "Recently, it has been found that myosin VI regulates fusion pores formed between secretory vesicles and the plasma membrane [25]. In contrast to myosin Vb and Vc, myosin VI was present in human, wild-type and Myo5a−/− platelets. "
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    ABSTRACT: The motor protein myosin Va plays an important role in the trafficking of intracellular vesicles. Mutation of the Myo5a gene causes Griscelli syndrome type 1 in humans and the dilute phenotype in mice, which are both characterised by pigment dilution and neurological defects as a result of impaired vesicle transport in melanocytes and neuroendocrine cells. The role of myosin Va in platelets is currently unknown. Rab27 has been shown to be associated with myosin Va cargo vesicles and is known to be important in platelet dense granule biogenesis and secretion, a crucial event in thrombus formation. Therefore, we hypothesised that myosin Va may regulate granule secretion or formation in platelets. Platelet function was studied in vitro using a novel Myo5a gene deletion mouse model. Myo5a(-/-) platelets were devoid of myosin Va, as determined by immunoblotting, and exhibited normal expression of surface markers. We assessed dense granule, α-granule and lysosomal secretion, integrin α(IIb)β(3) activation, Ca(2+) signalling, and spreading on fibrinogen in response to collagen-related peptide or the PAR4 agonist, AYPGKF in washed mouse platelets lacking myosin Va or wild-type platelets. Surprisingly, Myo5a(-/-) platelets showed no significant functional defects in these responses, or in the numbers of dense and α-granules expressed. Despite the importance of myosin Va in vesicle transport in other cells, our data demonstrate this motor protein has no non-redundant role in the secretion of dense and α-granules or other functional responses in platelets.
    Full-text · Article · Jan 2013 · PLoS ONE
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    • "As a component of the TNFR1 complex, optineurin is furthermore involved in regulation of NF-κB signaling by competing with IKKγ for ubiquitinated RIP1 [11], [17]. In addition, a role for optineurin in endo- and exocytotic vesicle trafficking has been demonstrated [12], [18]. "
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    ABSTRACT: Escape from the host immune system is essential for intracellular pathogens. The adenoviral protein E3-14.7K (14.7K) is known as a general inhibitor of tumor necrosis factor (TNF)-induced apoptosis. It efficiently blocks TNF-receptor 1 (TNFR1) internalization but the underlying molecular mechanism still remains elusive. Direct interaction of 14.7K and/or associated proteins with the TNFR1 complex has been discussed although to date not proven. In our study, we provide for the first time evidence for recruitment of 14.7K and the 14.7K interacting protein optineurin to TNFR1. Various functions have been implicated for optineurin such as regulation of receptor endocytosis, vesicle trafficking, regulation of the nuclear factor κB (NF-κB) pathway and antiviral signaling. We therefore hypothesized that binding of optineurin to 14.7K and recruitment of both proteins to the TNFR1 complex is essential for protection against TNF-induced cytotoxic effects. To precisely dissect the individual role of 14.7K and optineurin, we generated and characterized a 14.7K mutant that does not confer TNF-resistance but is still able to interact with optineurin. In H1299 and KB cells expressing 14.7K wild-type protein, neither decrease in cell viability nor cleavage of caspases was observed upon stimulation with TNF. In sharp contrast, cells expressing the non-protective mutant of 14.7K displayed reduced viability and cleavage of initiator and effector caspases upon TNF treatment, indicating ongoing apoptotic cell death. Knockdown of optineurin in 14.7K expressing cells did not alter the protective effect as measured by cell viability and caspase activation. Taken together, we conclude that optineurin despite its substantial role in vesicular trafficking, endocytosis of cell surface receptors and recruitment to the TNFR1 complex is dispensable for the 14.7K-mediated protection against TNF-induced apoptosis.
    Full-text · Article · Jun 2012 · PLoS ONE
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