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Thyroid cancer has the fastest growing incidence of any cancer in the United States, as measured by the number of new cases per year. Despite advances in tissue culture techniques, a robust model for thyroid cancer spheroid culture is yet to be developed. Using eight established thyroid cancer cell lines, we created an efficient and cost-effective 3D culture system that can enhance our understanding of in vivo treatment response. We found that all eight cell lines readily form spheroids in culture with unique morphology, size, and cytoskeletal organization. In addition, we developed a high-throughput workflow that allows for drug screening of spheroids. Using this approach, we found that spheroids from K1 and TPC1 cells demonstrate significant differences in their sensitivities to dabrafenib treatment that closely model expected patient drug response. In addition, K1 spheroids have increased sensitivity to dabrafenib when compared to monolayer K1 cultures. Utilizing traditional 2D cultures of these cell lines, we evaluated the mechanisms of this drug response, showing dramatic and acute changes in their actin cytoskeleton as well as inhibition of migratory behavior in response to dabrafenib treatment. Our study is the first to describe the development of a robust spheroid system from established cultured thyroid cancer cell lines and adaptation to a high-throughput format. We show that combining 3D culture with traditional 2D methods provides a complementary and powerful approach to uncover drug sensitivity and mechanisms of inhibition in thyroid cancer.

Dendrite growth is constrained by a self-avoidance response that induces retraction but the downstream pathways that balance these opposing mechanisms are unknown. We have proposed that the diffusible cue UNC-6(Netrin) is captured by UNC-40(DCC) for a short-range interaction with UNC-5 to trigger self-avoidance in the C. elegans PVD neuron. Here we report that the actin-polymerizing proteins UNC-34(Ena/VASP), WSP-1(WASP), UNC-73(Trio), MIG-10(Lamellipodin) and the Arp2/3 complex effect dendrite retraction in the self-avoidance response mediated by UNC-6(Netrin). The paradoxical idea that actin polymerization results in shorter rather than longer dendrites is explained by our finding that NMY-1 (non-muscle myosin II) is necessary for retraction and could therefore mediate this effect in a contractile mechanism. Our results also show that dendrite length is determined by the antagonistic effects on the actin cytoskeleton of separate sets of effectors for retraction mediated by UNC-6(Netrin) versus outgrowth promoted by the DMA-1 receptor. Thus, our findings suggest that the dendrite length depends on an intrinsic mechanism that balances distinct modes of actin assembly for growth versus retraction.

Animal cells, amoebas and yeast divide using a force-generating, actin- and myosin-based contractile ring or 'cytokinetic ring' (CR). Despite intensive research, questions remain about the spatial organization of CR components, the mechanism by which the CR generates force, and how other cellular processes are coordinated with the CR for successful membrane ingression and ultimate cell separation. This Review highlights new findings about the spatial relationship of the CR to the plasma membrane and the arrangement of molecules within the CR from studies using advanced microscopy techniques, as well as mechanistic information obtained from approaches. We also consider advances in understanding coordinated cellular processes that impact the architecture and function of the CR.
© 2019. Published by The Company of Biologists Ltd.

In animals and fungi, cytokinesis is facilitated by the constriction of an actomyosin contractile ring (CR) [1]. In Schizosaccharomyces pombe, the CR forms mid-cell during mitosis from clusters of proteins at the medial cell cortex called nodes [2]. The anillin-like protein Mid1 localizes to nodes and is required for CR assembly at mid-cell [3]. When CR constriction begins, Mid1 leaves the division site. How Mid1 disassociates and whether this step is important for cytokinetic progression has been unknown. The septation initiation network (SIN), analogous to the Hippo pathway of multicellular organisms, is a signaling cascade that triggers node dispersal, CR assembly and constriction, and septum formation [4, 5]. We report that the terminal SIN kinase, Sid2 [6], phosphorylates Mid1 to drive its removal from the cortex at CR constriction onset. A Mid1 mutant that cannot be phosphorylated by Sid2 remains cortical during cytokinesis, over-accumulates in interphase nodes following cell division in a manner dependent on the SAD kinase Cdr2, advances the G2/M transition, precociously recruits other CR components to nodes, pulls Cdr2 aberrantly into the CR, and reduces rates of CR maturation and constriction. When combined with cdr2 mutants that affect node assembly or disassembly, gross defects in division site positioning result. Our findings identify Mid1 as a key Sid2 substrate for SIN-mediated remodeling of the division site for efficient cytokinesis and provide evidence that nodes serve to integrate signals coordinating cell cycle progression and cytokinesis.
Copyright © 2019 Elsevier Ltd. All rights reserved.

The sarcomere is the contractile unit within cardiomyocytes driving heart muscle contraction. We sought to test the mechanisms regulating actin and myosin filament assembly during sarcomere formation. Therefore, we developed an assay using human cardiomyocytes to monitor sarcomere assembly. We report a population of muscle stress fibers, similar to actin arcs in non-muscle cells, which are essential sarcomere precursors. We show sarcomeric actin filaments arise directly from muscle stress fibers. This requires formins (e.g., FHOD3), non-muscle myosin IIA and non-muscle myosin IIB. Furthermore, we show short cardiac myosin II filaments grow to form ~1.5 μm long filaments that then 'stitch' together to form the stack of filaments at the core of the sarcomere (i.e., the A-band). A-band assembly is dependent on the proper organization of actin filaments and, as such, is also dependent on FHOD3 and myosin IIB. We use this experimental paradigm to present evidence for a unifying model of sarcomere assembly.
© 2018, Fenix et al.

IMPACT STATEMENT - Subarachnoid hemorrhage (SAH) is associated with vasospasm that is refractory to traditional vasodilators, and inhibition of vasospasm after SAH remains a large unmet clinical need. SAH causes changes in the phosphorylation state of the small heat shock proteins (HSPs), HSP20 and HSP27, in the vasospastic vessels. In this study, the levels of HSP27 and HSP20 were manipulated using nanotechnology to mimic the intracellular phenotype of SAH-induced vasospasm, and the effect of this manipulation was tested on vasomotor responses in intact tissues. This work provides insight into potential therapeutic targets for the development of more effective treatments for SAH induced vasospasm.

How cellular contractile systems assemble has fascinated scientists for generations. The major molecule responsible for cellular force generation is the molecular motor, non-muscle myosin II (NMII). NMII molecules are organized into single myosin filaments and larger arrays of filaments called NMII stacks, which are capable of generating increasing amounts of force. The textbook model of NMII stack assembly is the Network Contraction Model, where ensembles of distinct NMII filaments condense into a NMII stack by pulling on actin filaments. While this model has been widely accepted for ~20 years, it has been difficult to test inside cells due to the small size of NMII filaments. Recently, interest in how NMII stacks form has been reinvigorated by the advent of super-resolution microscopy techniques which have afforded unprecedented resolution of NMII filaments inside cells. A number of recent publications using these techniques have called into question key aspects of the Network Contraction Model, and our understanding of how NMII stacks assemble.
© 2018 Wiley Periodicals, Inc.

Proper morphogenesis of dendrites plays a fundamental role in the establishment of neural circuits. The molecular mechanism by which dendrites grow highly complex branches is not well understood. Here, using the Caenorhabditis elegans PVD neuron, we demonstrate that high-order dendritic branching requires actin polymerization driven by coordinated interactions between two membrane proteins, DMA-1 and HPO-30, with their cytoplasmic interactors, the RacGEF TIAM-1 and the actin nucleation promotion factor WAVE regulatory complex (WRC). The dendrite branching receptor DMA-1 directly binds to the PDZ domain of TIAM-1, while the claudin-like protein HPO-30 directly interacts with the WRC. On dendrites, DMA-1 and HPO-30 form a receptor-associated signaling complex to bring TIAM-1 and the WRC to close proximity, leading to elevated assembly of F-actin needed to drive high-order dendrite branching. The synergistic activation of F-actin assembly by scaffolding distinct actin regulators might represent a general mechanism in promoting complex dendrite arborization.
Copyright © 2018. Published by Elsevier Inc.

In , cytokinesis requires the assembly and constriction of an actomyosin-based contractile ring (CR). A single essential formin, Cdc12, localizes to the cell middle upon mitotic onset and nucleates the F-actin of the CR. Cdc12 medial recruitment is mediated in part by its direct binding to the F-BAR scaffold Cdc15. Given that Cdc12 is hyperphosphorylated in M phase, we explored whether Cdc12 phosphoregulation impacts its association with Cdc15 during mitosis. We found that Cdk1, a major mitotic kinase, phosphorylates Cdc12 on six N-terminal residues near the Cdc15-binding site, and phosphorylation on these sites inhibits its interaction with the Cdc15 F-BAR domain. Consistent with this finding, a mutant with all six Cdk1 sites changed to phosphomimetic residues () displays phenotypes similar to , in which the Cdc15-binding motif is disrupted; both show reduced Cdc12 at the CR and delayed CR formation. Together, these results indicate that Cdk1 phosphorylation of formin Cdc12 antagonizes its interaction with Cdc15 and thereby opposes Cdc12's CR localization. These results are consistent with a general role for Cdk1 in inhibiting cytokinesis until chromosome segregation is complete.
© 2018 Willet et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Arrestins recruit a variety of signaling proteins to active phosphorylated G protein-coupled receptors in the plasma membrane and to the cytoskeleton. Loss of arrestins leads to decreased cell migration, altered cell shape, and an increase in focal adhesions. Small GTPases of the Rho family are molecular switches that regulate actin cytoskeleton and affect a variety of dynamic cellular functions including cell migration and cell morphology. Here we show that non-visual arrestins differentially regulate RhoA and Rac1 activity to promote cell spreading via actin reorganization, and focal adhesion formation via two distinct mechanisms. Arrestins regulate these small GTPases independently of G-protein-coupled receptor activation.
Copyright © 2017 Elsevier Inc. All rights reserved.