GLUT1CBP(TIP2/GIPC1) interactions with GLUT1 and myosin VI: evidence supporting an adapter function for GLUT1CBP

We identified a novel interaction between myosin VI and the GLUT1 transporter binding protein GLUT1CBP(GIPC1) and first proposed that as an adapter molecule it might function to couple vesicle-bound proteins to myosin VI movement. This study refines the model by identifying two myosin VI binding domains in the GIPC1 C terminus, assigning respective oligomerization and myosin VI binding functions to separate N- and C-terminal domains, and defining a central region in the myosin VI tail that binds GIPC1. Data further supporting the model demonstrate that 1) myosin VI and GIPC1 interactions do not require a mediating protein; 2) the myosin VI binding domain in GIPC1 is necessary for intracellular interactions of GIPC1 with myosin VI and recruitment of overexpressed myosin VI to membrane structures, but not for the association of GIPC1 with such structures; 3) GIPC1/myosin VI complexes coordinately move within cellular extensions of the cell in an actin-dependent and microtubule-independent manner; and 4) blocking either GIPC1 interactions with myosin VI or GLUT1 interactions with GIPC1 disrupts normal GLUT1 trafficking in polarized epithelial cells, leading to a reduction in the level of GLUT1 in the plasma membrane and concomitant accumulation in internal membrane structures.     

Myosin VI targeting to clathrin-coated structures and dimerization is mediated by binding to Disabled-2 and PtdIns(4,5)P2

Vesicle transport is essential for the movement of proteins, lipids and other molecules between membrane compartments within the cell. The role of the class VI myosins in vesicular transport is particularly intriguing because they are the only class that has been shown to move 'backwards' towards the minus end of actin filaments. Myosin VI is found in distinct intracellular locations and implicated in processes such as endocytosis, exocytosis, maintenance of Golgi morphology and cell movement. We have shown that the carboxy-terminal tail is the key targeting region and have identified three binding sites: a WWY motif for Disabled-2 (Dab2) binding, a RRL motif for glucose-transporter binding protein (GIPC) and optineurin binding and a site that binds specifically and with high affinity (Kd = 0.3 microM) to PtdIns(4,5)P2-containing liposomes. This is the first demonstration that myosin VI binds lipid membranes. Lipid binding induces a large structural change in the myosin VI tail (31% increase in helicity) and when associated with lipid vesicles, it can dimerize. In vivo targeting and recruitment of myosin VI to clathrin-coated structures (CCSs) at the plasma membrane is mediated by Dab2 and PtdIns(4,5)P2 binding.     

Myosin VI is required for sorting of AP-1B-dependent cargo to the basolateral domain in polarized MDCK cells

In polarized epithelial cells, newly synthesized membrane proteins are delivered on specific pathways to either the apical or basolateral domains, depending on the sorting motifs present in these proteins. Because myosin VI has been shown to facilitate secretory traffic in nonpolarized cells, we investigated its role in biosynthetic trafficking pathways in polarized MDCK cells. We observed that a specific splice isoform of myosin VI with no insert in the tail domain is required for the polarized transport of tyrosine motif containing basolateral membrane proteins. Sorting of other basolateral or apical cargo, however, does not involve myosin VI. Site-directed mutagenesis indicates that a functional complex consisting of myosin VI, optineurin, and probably the GTPase Rab8 plays a role in the basolateral delivery of membrane proteins, whose sorting is mediated by the clathrin adaptor protein complex (AP) AP-1B. Our results suggest that myosin VI is a crucial component in the AP-1B-dependent biosynthetic sorting pathway to the basolateral surface in polarized epithelial cells.     

Multimodal regulation of myosin VI ensemble transport by cargo adaptor protein GIPC

A range of cargo adaptor proteins are known to recruit cytoskeletal motors to distinct subcellular compartments. However, the structural impact of cargo recruitment on motor function is poorly understood. Here, we dissect the multimodal regulation of myosin VI activity through the cargo adaptor GAIP-interacting protein, C terminus (GIPC), whose overexpression with this motor in cancer enhances cell migration. Using a range of biophysical techniques, including motility assays, FRET-based conformational sensors, optical trapping, and DNA origami–based cargo scaffolds to probe the individual and ensemble properties of GIPC–myosin VI motility, we report that the GIPC myosin-interacting region (MIR) releases an autoinhibitory interaction within myosin VI. We show that the resulting conformational changes in the myosin lever arm, including the proximal tail domain, increase the flexibility of the adaptor–motor linkage, and that increased flexibility correlates with faster actomyosin association and dissociation rates. Taken together, the GIPC MIR–myosin VI interaction stimulates a twofold to threefold increase in ensemble cargo speed. Furthermore, the GIPC MIR–myosin VI ensembles yield similar cargo run lengths as forced processive myosin VI dimers. We conclude that the emergent behavior from these individual aspects of myosin regulation is the fast, processive, and smooth cargo transport on cellular actin networks. Our study delineates the multimodal regulation of myosin VI by the cargo adaptor GIPC, while highlighting linkage flexibility as a novel biophysical mechanism for modulating cellular cargo motility.     

Supervillin binding to myosin II and synergism with anillin are required for cytokinesis

Cytokinesis, the process by which cytoplasm is apportioned between dividing daughter cells, requires coordination of myosin II function, membrane trafficking, and central spindle organization. Most known regulators act during late cytokinesis; a few, including the myosin II–binding proteins anillin and supervillin, act earlier. Anillin''s role in scaffolding the membrane cortex with the central spindle is well established, but the mechanism of supervillin action is relatively uncharacterized. We show here that two regions within supervillin affect cell division: residues 831–1281, which bind central spindle proteins, and residues 1–170, which bind the myosin II heavy chain (MHC) and the long form of myosin light-chain kinase. MHC binding is required to rescue supervillin deficiency, and mutagenesis of this site creates a dominant-negative phenotype. Supervillin concentrates activated and total myosin II at the furrow, and simultaneous knockdown of supervillin and anillin additively increases cell division failure. Knockdown of either protein causes mislocalization of the other, and endogenous anillin increases upon supervillin knockdown. Proteomic identification of interaction partners recovered using a high-affinity green fluorescent protein nanobody suggests that supervillin and anillin regulate the myosin II and actin cortical cytoskeletons through separate pathways. We conclude that supervillin and anillin play complementary roles during vertebrate cytokinesis.     

Myosin II regulatory light chain as a novel substrate for AIM-1, an aurora/Ipl1p-related kinase from rat

Previous studies demonstrated that the phosphorylated myosin II regulatory light chain (MRLC) is localized at the cleavage furrow of dividing cells, suggesting that phosphorylation of MRLC plays an important role in cytokinesis. However, it remains unclear which kinase(s) phosphorylate MRLC during cytokinesis. AIM-1, an Aurora/Ipl1p-related kinase from rat, is known as a serine/threonine kinase that is required for cytokinesis. Here we examined the possibility that AIM-1 is a candidate for a kinase that phosphorylates MRLC during cytokinesis. As a result, we showed that AIM-1 monophosphorylated MRLC at Ser19 using two-dimensional phosphopeptide mapping analysis and several MRLC mutants. Furthermore, AIM-1 was colocalized with monophosphorylated MRLC at the cleavage furrow of dividing cells. We propose here that AIM-1 may participate in monophosphorylation of MRLC during cytokinesis.     

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

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.     

Cortical actin turnover during cytokinesis requires myosin II

The involvement of myosin II in cytokinesis has been demonstrated with microinjection, genetic, and pharmacological approaches; however, the exact role of myosin II in cell division remains poorly understood. To address this question, we treated dividing normal rat kidney (NRK) cells with blebbistatin, a potent inhibitor of the nonmuscle myosin II ATPase. Blebbistatin caused a strong inhibition of cytokinesis but no detectable effect on the equatorial localization of actin or myosin. However, whereas these filaments dissociated from the equator in control cells during late cytokinesis, they persisted in blebbistatin-treated cells over an extended period of time. The accumulation of equatorial actin was caused by the inhibition of actin filament turnover, as suggested by a 2-fold increase in recovery half-time after fluorescence photobleaching. Local release of blebbistatin at the equator caused localized accumulation of equatorial actin and inhibition of cytokinesis, consistent with the function of myosin II along the furrow. However, treatment of the polar region also caused a high frequency of abnormal cytokinesis, suggesting that myosin II may play a second, global role. Our observations indicate that myosin II ATPase is not required for the assembly of equatorial cortex during cytokinesis but is essential for its subsequent turnover and remodeling.     

Biphasic targeting and cleavage furrow ingression directed by the tail of a myosin II

Cytokinesis in animal and fungal cells utilizes a contractile actomyosin ring (AMR). However, how myosin II is targeted to the division site and promotes AMR assembly, and how the AMR coordinates with membrane trafficking during cytokinesis, remains poorly understood. Here we show that Myo1 is a two-headed myosin II in Saccharomyces cerevisiae, and that Myo1 localizes to the division site via two distinct targeting signals in its tail that act sequentially during the cell cycle. Before cytokinesis, Myo1 localization depends on the septin-binding protein Bni5. During cytokinesis, Myo1 localization depends on the IQGAP Iqg1. We also show that the Myo1 tail is sufficient for promoting the assembly of a "headless" AMR, which guides membrane deposition and extracellular matrix remodeling at the division site. Our study establishes a biphasic targeting mechanism for myosin II and highlights an underappreciated role of the AMR in cytokinesis beyond force generation.     

Localization of PKA phosphorylation site, Ser(2030), in the three-dimensional structure of cardiac ryanodine receptor

Cofilin regulates actin filament dynamics by stimulating actin filament disassembly and plays a critical role in cytokinesis and chemotactic migration. Aip1 (actin-interacting protein 1), also called WDR1 (WD-repeat protein 1), is a highly conserved WD-repeat protein in eukaryotes and promotes cofilin-mediated actin filament disassembly in vitro; however, little is known about the mechanisms by which Aip1 functions in cytokinesis and cell migration in mammalian cells. In the present study, we investigated the roles of Aip1 in cytokinesis and chemotactic migration of human cells by silencing the expression of Aip1 using siRNA (small interfering RNA). Knockdown of Aip1 in HeLa cells increased the percentage of multinucleate cells; this effect was reversed by expression of an active form of cofilin. In Aip1-knockdown cells, the cleavage furrow ingressed normally from anaphase to early telophase; however, an excessive accumulation of actin filaments was observed on the contractile ring in late telophase. These results suggest that Aip1 plays a crucial role in the completion of cytokinesis by promoting cofilin-mediated actin filament disassembly in telophase. We have also shown that Aip1 knockdown significantly suppressed chemokine-induced chemotactic migration of Jurkat T-lymphoma cells, and this was blocked by expression of an active form of cofilin. Whereas control cells mostly formed a single lamellipodium in response to chemokine stimulation, Aip1 knockdown cells abnormally exhibited multiple protrusions around the cells before and after cell stimulation. This indicates that Aip1 plays an important role in directional cell migration by restricting the stimulus-induced membrane protrusion to one direction via promoting cofilin activity.     

A specific isoform of nonmuscle myosin II-C is required for cytokinesis in a tumor cell line

Nonmuscle myosin IIs play an essential role during cytokinesis. Here, we explore the function of an alternatively spliced isoform of nonmuscle myosin heavy chain (NMHC) II-C, called NMHC II-C1, in the A549 human lung tumor cell line during cytokinesis. NMHC II-C1 contains an insert of 8 amino acids in the head region of NMHC II-C. First, we show that there is a marked increase in both the mRNA encoding NMHC II-C1 and protein in tumor cell lines compared with nontumor cell lines derived from the same tissue. Quantification of the amount of myosin II isoforms in the A549 cells shows that the amounts of NMHC II-A and II-C1 protein are about equal and substantially greater than NMHC II-B. Using specific siRNAs to decrease NMHC II-C1 in cultured A549 cells resulted in a 5.5-fold decrease in the number of cells at 120 h, whereas decreasing NMHC II-A with siRNA does not affect cell proliferation. This decreased proliferation can be rescued by reintroducing NMHC II-C1 but not NMHC II-A or II-B into A549 cells, although noninserted NMHC II-C does rescue to a limited extent. Time lapse video microscopy revealed that loss of NMHC II-C1 leads to a delay in cytokinesis and prolongs it from 2 to 8-10 h. These findings are consistent with the localization of NMHC II-C1 to the intercellular bridge that attaches the two dividing cells during the late phases of cytokinesis. The results suggest a specific function for NMHC II-C1 in cytokinesis in the A549 tumor cell line.     

Mammalian SEPT2 is required for scaffolding nonmuscle myosin II and its kinases

Mammalian septin SEPT2 belongs to a conserved family of filamentous GTPases that are associated with actin stress fibers in interphase cells and the contractile ring in dividing cells. Although SEPT2 is essential for cytokinesis, its role in this process remains undefined. Here, we report that SEPT2 directly binds nonmuscle myosin II (myosin II), and this association is important for fully activating myosin II in interphase and dividing cells. Inhibition of the SEPT2-myosin II interaction in interphase cells results in loss of stress fibers, while in dividing cells this causes instability of the ingressed cleavage furrow and dissociation of the myosin II from the Rho-activated myosin kinases ROCK and citron kinase. We propose that SEPT2-containing filaments provide a molecular platform for myosin II and its kinases to ensure the full activation of myosin II that is necessary for the final stages of cytokinesis.     

Anillin binds nonmuscle myosin II and regulates the contractile ring

We demonstrate that the contractile ring protein anillin interacts directly with nonmuscle myosin II and that this interaction is regulated by myosin light chain phosphorylation. We show that despite their interaction, anillin and myosin II are independently targeted to the contractile ring. Depletion of anillin in Drosophila or human cultured cells results in cytokinesis failure. Human cells depleted for anillin fail to properly regulate contraction by myosin II late in cytokinesis and fail in abscission. We propose a role for anillin in spatially regulating the contractile activity of myosin II during cytokinesis.     

Unconventional Myosin ID is Involved in Remyelination After Cuprizone-Induced Demyelination

Myelin, which is a multilamellar structure that sheathes the axon, is essential for normal neuronal function. In the central nervous system (CNS), myelin is produced by oligodendrocytes (OLs), which wrap their plasma membrane around axons. The dynamic membrane trafficking system, which relies on motor proteins, is required for myelin formation and maintenance. Previously, we reported that myosin ID (Myo1d) is distributed in rat CNS myelin and is especially enriched in the outer and inner cytoplasm-containing loops. Further, small interfering RNA (siRNA) treatment highlighted the involvement of Myo1d in the formation and maintenance of myelin in cultured OLs. Myo1d is one of the unconventional myosins, which may contribute to membrane dynamics, either in the wrapping process or transport of myelin membrane proteins during myelination. However, the function of Myo1d in myelin formation in vivo remains unclear. In the current study, to clarify the function of Myo1d in vivo, we surgically injected siRNA in the corpus callosum of a cuprizone-treated demyelination mouse model via stereotaxy. Knockdown of Myo1d expression in vivo decreased the intensities of myelin basic protein and myelin proteolipid protein immunofluorescence staining. However, neural/glial antigen 2-positive signals and adenomatous polyposis coli (APC/CC1)-positive cell numbers were unchanged by siRNA treatment. Furthermore, Myo1d knockdown treatment increased pro-inflammatory microglia and astrocytes during remyelination. In contrast, anti-inflammatory microglia were decreased. The percentage of caspase 3-positive cells in total CC1-positive OLs were also increased by Myo1d knockdown. These results indicated that Myo1d plays an important role during the regeneration process after demyelination.

     

Cytokinesis mediated through the recruitment of cortexillins into the cleavage furrow.

The fact that substrate-anchored Dictyostelium cells undergo cytokinesis in the absence of myosin II underscores the importance of other proteins in enabling the cleavage furrow to constrict. Cortexillins, a pair of actin-bundling proteins, are required for normal cleavage. They are targeted to the incipient furrow in wild-type and, more prominently, in myosin II-null cells. No other F-actin bundling or cross-linking protein tested is co-localized. Green fluorescent protein fusions show that the N-terminal actin-binding domain of cortexillin I is dispensable and the C-terminal region is sufficient for translocation to the furrow and the rescue of cytokinesis. Cortexillins are suggested to have a targeting signal for coupling to a myosin II-independent system that directs transport of membrane proteins to the cleavage furrow.     

Distinct pathways for the early recruitment of myosin II and actin to the cytokinetic furrow

Equatorial organization of myosin II and actin has been recognized as a universal event in cytokinesis of animal cells. Current models for the formation of equatorial cortex favor either directional cortical transport toward the equator or localized de novo assembly. However, this process has never been analyzed directly in dividing mammalian cells at a high resolution. Here we applied total internal reflection fluorescence microscope (TIRF-M), coupled with spatial temporal image correlation spectroscopy (STICS) and a new analytical approach termed temporal differential microscopy (TDM), to image the dynamics of myosin II and actin during the assembly of equatorial cortex. Our results indicated distinct and at least partially independent mechanisms for the early equatorial recruitment of myosin and actin filaments. Cortical myosin showed no detectable directional flow during early cytokinesis. In addition to equatorial assembly, we showed that localized inhibition of disassembly contributed to the formation of the equatorial myosin band. In contrast to myosin, actin filaments underwent a striking flux toward the equator. Myosin motor activity was required for the actin flux, but not for actin concentration in the furrow, suggesting that there was a flux-independent, de novo mechanism for actin recruitment along the equator. Our results indicate that cytokinesis involves signals that regulate both assembly and disassembly activities and argue against mechanisms that are coupled to global cortical movements.     

Myosin VI is a mediator of the p53-dependent cell survival pathway

Myosin VI is an unconventional motor protein, and its mutation is responsible for the familiar conditions sensorineural deafness and hypertrophic cardiomyopathy. Myosin VI is found to play a key role in the protein trafficking and homeostasis of the Golgi complex. However, very little is known about how myosin VI is regulated and whether myosin VI has a function in the DNA damage response. Here, we found that myosin VI is regulated by DNA damage in a p53-dependent manner and possesses a novel function in the p53-dependent prosurvival pathway. Specifically, we show that myosin VI is induced by p53 and DNA damage in a p53-dependent manner. We found that p53 directly binds to, and activates, the promoter of the myosin VI gene. We also show that the intracellular localization of myosin VI is substantially altered by p53 and DNA damage in a p53-dependent manner such that the pool of myosin VI in endocytic vesicles, membrane ruffles, and cytosol migrates to the Golgi complex, perinuclear membrane, and nucleus. Furthermore, we show that knockdown of myosin VI attenuates activation of p53 and impairs Golgi complex integrity, which makes myosin VI-deficient cells susceptible to apoptosis upon DNA damage. Taken together, we found a novel function for p53 in the maintenance of Golgi complex integrity and for myosin VI in the p53-dependent prosurvival pathway.     

Optineurin links myosin VI to the Golgi complex and is involved in Golgi organization and exocytosis

Myosin VI plays a role in the maintenance of Golgi morphology and in exocytosis. In a yeast 2-hybrid screen we identified optineurin as a binding partner for myosin VI at the Golgi complex and confirmed this interaction in a range of protein interaction studies. Both proteins colocalize at the Golgi complex and in vesicles at the plasma membrane. When optineurin is depleted from cells using RNA interference, myosin VI is lost from the Golgi complex, the Golgi is fragmented and exocytosis of vesicular stomatitis virus G-protein to the plasma membrane is dramatically reduced. Two further binding partners for optineurin have been identified: huntingtin and Rab8. We show that myosin VI and Rab8 colocalize around the Golgi complex and in vesicles at the plasma membrane and overexpression of constitutively active Rab8-Q67L recruits myosin VI onto Rab8-positive structures. These results show that optineurin links myosin VI to the Golgi complex and plays a central role in Golgi ribbon formation and exocytosis.     

Non-muscle myosin II regulates aortic stiffness through effects on specific focal adhesion proteins and the non-muscle cortical cytoskeleton

Non-muscle myosin II (NMII) plays a role in many fundamental cellular processes including cell adhesion, migration, and cytokinesis. However, its role in mammalian vascular function is not well understood. Here, we investigated the function of NMII in the biomechanical and signalling properties of mouse aorta. We found that blebbistatin, an inhibitor of NMII, decreases agonist-induced aortic stress and stiffness in a dose-dependent manner. We also specifically demonstrate that in freshly isolated, contractile, aortic smooth muscle cells, the non-muscle myosin IIA (NMIIA) isoform is associated with contractile filaments in the core of the cell as well as those in the non-muscle cell cortex. However, the non-muscle myosin IIB (NMIIB) isoform is excluded from the cell cortex and colocalizes only with contractile filaments. Furthermore, both siRNA knockdown of NMIIA and NMIIB isoforms in the differentiated A7r5 smooth muscle cell line and blebbistatin-mediated inhibition of NM myosin II suppress agonist-activated increases in phosphorylation of the focal adhesion proteins FAK Y925 and paxillin Y118. Thus, we show in the present study, for the first time that NMII regulates aortic stiffness and stress and that this regulation is mediated through the tension-dependent phosphorylation of the focal adhesion proteins FAK and paxillin.     

Myosin Heavy Chain Phosphorylation Sites Regulate Myosin Localization during Cytokinesis in Live Cells

Conventional myosin II plays a fundamental role in the process of cytokinesis where, in the form of bipolar thick filaments, it is thought to be the molecular motor that generates the force necessary to divide the cell. In Dictyostelium, the formation of thick filaments is regulated by the phosphorylation of three threonine residues in the tail region of the myosin heavy chain. We report here on the effects of this regulation on the localization of myosin in live cells undergoing cytokinesis. We imaged fusion proteins of the green-fluorescent protein with wild-type myosin and with myosins where the three critical threonines had been changed to either alanine or aspartic acid. We provide evidence that thick filament formation is required for the accumulation of myosin in the cleavage furrow and that if thick filaments are overproduced, this accumulation is markedly enhanced. This suggests that myosin localization in dividing cells is regulated by myosin heavy chain phosphorylation.