A proliferative to invasive switch is mediated by srGAP1 downregulation through the activation of TGF-ß2 signaling.

Many breast cancer (BC) patients suffer from complications of metastatic disease. To form metastases, cancer cells must become migratory and coordinate both invasive and proliferative programs at distant organs. Here, we identify srGAP1 as a regulator of a proliferative-to-invasive switch in BC cells. High-resolution light-sheet microscopy demonstrates that BC cells can form actin-rich protrusions during extravasation. srGAP1low cells display a motile and invasive phenotype that facilitates their extravasation from blood vessels, as shown in zebrafish and mouse models, while attenuating tumor growth. Interestingly, a population of srGAP1low cells remain as solitary disseminated tumor cells in the lungs of mice bearing BC tumors. Overall, srGAP1low cells have increased Smad2 activation and TGF-ß2 secretion, resulting in increased invasion and p27 levels to sustain quiescence. These findings identify srGAP1 as a mediator of a proliferative to invasive phenotypic switch in BC cells in vivo through a TGF-ß2-mediated signaling axis.

The SWI/SNF chromatin remodeling assemblies BAF and PBAF differentially regulate cell cycle exit and cellular invasion in vivo.

Chromatin remodelers such as the SWI/SNF complex coordinate metazoan development through broad regulation of chromatin accessibility and transcription, ensuring normal cell cycle control and cellular differentiation in a lineage-specific and temporally restricted manner. Mutations in genes encoding the structural subunits of chromatin, such as histone subunits, and chromatin regulating factors are associated with a variety of disease mechanisms including cancer metastasis, in which cancer co-opts cellular invasion programs functioning in healthy cells during development. Here we utilize Caenorhabditis elegans anchor cell (AC) invasion as an in vivo model to identify the suite of chromatin agents and chromatin regulating factors that promote cellular invasiveness. We demonstrate that the SWI/SNF ATP-dependent chromatin remodeling complex is a critical regulator of AC invasion, with pleiotropic effects on both G0 cell cycle arrest and activation of invasive machinery. Using targeted protein degradation and enhanced RNA interference (RNAi) vectors, we show that SWI/SNF contributes to AC invasion in a dose-dependent fashion, with lower levels of activity in the AC corresponding to aberrant cell cycle entry and increased loss of invasion. Our data specifically implicate the SWI/SNF BAF assembly in the regulation of the G0 cell cycle arrest in the AC, whereas the SWI/SNF PBAF assembly promotes AC invasion via cell cycle-independent mechanisms, including attachment to the basement membrane (BM) and activation of the pro-invasive fos-1/FOS gene. Together these findings demonstrate that the SWI/SNF complex is necessary for two essential components of AC invasion: arresting cell cycle progression and remodeling the BM. The work here provides valuable single-cell mechanistic insight into how the SWI/SNF assemblies differentially contribute to cellular invasion and how SWI/SNF subunit-specific disruptions may contribute to tumorigeneses and cancer metastasis.

Stu2 uses a 15-nm parallel coiled coil for kinetochore localization and concomitant regulation of the mitotic spindle.

XMAP215/Dis1 family proteins are potent microtubule polymerases, critical for mitotic spindle structure and dynamics. While microtubule polymerase activity is driven by an N-terminal tumor overexpressed gene (TOG) domain array, proper cellular localization is a requisite for full activity and is mediated by a C-terminal domain. Structural insight into the C-terminal domain's architecture and localization mechanism remain outstanding. We present the crystal structure of the Saccharomyces cerevisiae Stu2 C-terminal domain, revealing a 15-nm parallel homodimeric coiled coil. The parallel architecture of the coiled coil has mechanistic implications for the arrangement of the homodimer's N-terminal TOG domains during microtubule polymerization. The coiled coil has two spatially distinct conserved regions: CRI and CRII. Mutations in CRI and CRII perturb the distribution and localization of Stu2 along the mitotic spindle and yield defects in spindle morphology including increased frequencies of mispositioned and fragmented spindles. Collectively, these data highlight roles for the Stu2 dimerization domain as a scaffold for factor binding that optimally positions Stu2 on the mitotic spindle to promote proper spindle structure and dynamics.

Biochemical and bioinformatic analysis of the myosin-XIX motor domain.

Mitochondrial dynamics are dependent on both the microtubule and actin cytoskeletal systems. Evidence for the involvement of myosin motors has been described in many systems, and until recently a candidate mitochondrial myosin transport motor had not been described in vertebrates. Myosin-XIX (MYO19) was predicted to represent a novel class of myosin and had previously been shown to bind to mitochondria and increase mitochondrial network dynamics when ectopically expressed. Our analyses comparing ∼40 MYO19 orthologs to ∼2000 other myosin motor domain sequences identified instances of homology well-conserved within class XIX myosins that were not found in other myosin classes, suggesting MYO19-specific mechanochemistry. Steady-state biochemical analyses of the MYO19 motor domain indicate that Homo sapiens MYO19 is a functional motor. Insect cell-expressed constructs bound calmodulin as a light chain at the predicted stoichiometry and displayed actin-activated ATPase activity. MYO19 constructs demonstrated high actin affinity in the presence of ATP in actin-co-sedimentation assays, and translocated actin filaments in gliding assays. Expression of GFP-MYO19 containing a mutation impairing ATPase activity did not enhance mitochondrial network dynamics, as occurs with wild-type MYO19, indicating that myosin motor activity is required for mitochondrial motility. The measured biochemical properties of MYO19 suggest it is a high-duty ratio motor that could serve to transport mitochondria or anchor mitochondria, depending upon the cellular microenvironment.

Human Myo19 is a novel myosin that associates with mitochondria.

Mitochondria are pleomorphic organelles that have central roles in cell physiology. Defects in their localization and dynamics lead to human disease. Myosins are actin-based motors that power processes such as muscle contraction, cytokinesis, and organelle transport. Here we report the initial characterization of myosin-XIX (Myo19), the founding member of a novel class of myosin that associates with mitochondria. The 970 aa heavy chain consists of a motor domain, three IQ motifs, and a short tail. Myo19 mRNA is expressed in multiple tissues, and antibodies to human Myo19 detect an approximately 109 kDa band in multiple cell lines. Both endogenous Myo19 and GFP-Myo19 exhibit striking localization to mitochondria. Deletion analysis reveals that the Myo19 tail is necessary and sufficient for mitochondrial localization. Expressing full-length GFP-Myo19 in A549 cells reveals a remarkable gain of function where the majority of the mitochondria move continuously. Moving mitochondria travel for many micrometers with an obvious leading end and distorted shape. The motility and shape change are sensitive to latrunculin B, indicating that both are actin dependent. Expressing the GFP-Myo19 tail in CAD cells resulted in decreased mitochondrial run lengths in neurites. These results suggest that this novel myosin functions as an actin-based motor for mitochondrial movement in vertebrate cells.