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Membrane blebs are specialized cellular protrusions that play diverse roles in processes such as cell division and cell migration. Blebbing can be divided into three distinct phases: bleb nucleation, bleb growth, and bleb retraction. Following nucleation and bleb growth, the actin cortex, comprising actin, cross-linking proteins, and nonmuscle myosin II (MII), begins to reassemble on the membrane. MII then drives the final phase, bleb retraction, which results in reintegration of the bleb into the cellular cortex. There are three MII paralogues with distinct biophysical properties expressed in mammalian cells: MIIA, MIIB, and MIIC. Here we show that MIIA specifically drives bleb retraction during cytokinesis. The motor domain and regulation of the nonhelical tailpiece of MIIA both contribute to its ability to drive bleb retraction. These experiments have also revealed a relationship between faster turnover of MIIA at the cortex and its ability to drive bleb retraction.
Intimal stiffening has been linked with increased vascular permeability and leukocyte transmigration, hallmarks of atherosclerosis. However, recent evidence indicates age-related intimal stiffening is not uniform but rather characterized by increased point-to-point heterogeneity in subendothelial matrix stiffness, the impact of which is much less understood. To investigate the impact of spatially heterogeneous matrix rigidity on endothelial monolayer integrity, we develop a micropillar model to introduce closely-spaced, step-changes in substrate rigidity and compare endothelial monolayer phenotype to rigidity-matched, uniformly stiff and compliant substrates. We found equivalent disruption of adherens junctions within monolayers on step-rigidity and uniformly stiff substrates relative to uniformly compliant substrates. Similarly, monolayers cultured on step-rigidity substrates exhibited equivalent percentages of leukocyte transmigration to monolayers on rigidity-matched, uniformly stiff substrates. Adherens junction tension and focal adhesion density, but not size, increased within monolayers on step-rigidity and uniformly stiff substrates compared to more compliant substrates suggesting that elevated tension is disrupting adherens junction integrity. Leukocyte transmigration frequency and time, focal adhesion size, and focal adhesion density did not differ between stiff and compliant sub-regions of step-rigidity substrates. Overall, our results suggest that endothelial monolayers exposed to mechanically heterogeneous substrates adopt the phenotype associated with the stiffer matrix, indicating that spatial heterogeneities in intimal stiffness observed with age could disrupt endothelial barrier integrity and contribute to atherogenesis.
During mouse pancreas organogenesis, endocrine cells are born from progenitors residing in an epithelial plexus niche. After a period in a lineage-primed state, progenitors become endocrine committed via upregulation of We find that the to transition is associated with distinct stages of an epithelial egression process: narrowing the apical surface of the cell, basalward cell movement and eventual cell-rear detachment from the apical lumen surface to allow clustering as nascent islets under the basement membrane. Apical narrowing, basalward movement and transcriptional upregulation still occur without Neurog3 protein, suggesting that morphogenetic cues deployed within the plexus initiate endocrine commitment upstream or independently of Neurog3. Neurog3 is required for cell-rear detachment and complete endocrine-cell birth. The ROCK-nmMyoII pathway coordinates epithelial-cell morphogenesis and the progression through -expressing states. NmMyoII is necessary for apical narrowing, basalward cell displacement and upregulation, but all three are limited by ROCK activity. We propose that ROCK-nmMyoII activity, gene-dose and Notch signaling integrate endocrine fate allocation with epithelial plexus growth and morphogenesis, representing a feedback control circuit that coordinates morphogenesis with lineage diversification in the endocrine-birth niche.
© 2018. Published by The Company of Biologists Ltd.
Metastasis is the most lethal aspect of cancer, yet current therapeutic strategies do not target its key rate-limiting steps. We have previously shown that the entry of cancer cells into the blood stream, or intravasation, is highly dependent upon in vivo cancer cell motility, making it an attractive therapeutic target. To systemically identify genes required for tumor cell motility in an in vivo tumor microenvironment, we established a novel quantitative in vivo screening platform based on intravital imaging of human cancer metastasis in ex ovo avian embryos. Utilizing this platform to screen a genome-wide shRNA library, we identified a panel of novel genes whose function is required for productive cancer cell motility in vivo, and whose expression is closely associated with metastatic risk in human cancers. The RNAi-mediated inhibition of these gene targets resulted in a nearly total (>99.5%) block of spontaneous cancer metastasis in vivo.
Cancer metastasis is the second leading cause of death in the United States. Despite its morbidity, metastasis is an inefficient process that few cells can survive. However, cancer cells can overcome these metastatic barriers via cellular responses to microenvironmental cues, such as through mechanotransduction. This review focuses on the mechanosensitive ion channels TRPV4 and P2X7, and their roles in metastasis, as both channels have been shown to significantly affect tumor cell dissemination. Upon activation, these channels help form tumor neovasculature, promote transendothelial migration, and increase cell motility. Conversely, they have also been linked to forms of cancer cell death dependent upon levels of activation, implying the complex functionality of mechanosensitive ion channels. Understanding the roles of TRPV4, P2X7 and other mechanosensitive ion channels in these processes may reveal new possible drug targets that modify channel function to reduce a tumor's metastatic potential.
Healthy pregnancy depends on proper placentation-including proliferation, differentiation, and invasion of trophoblast cells-which, if impaired, causes placental ischemia resulting in intrauterine growth restriction and preeclampsia. Mechanisms regulating trophoblast invasion, however, are unknown. We report that reduction of ( alters intracellular trafficking and significantly impairs invasion in a model of human extravillous trophoblasts. Furthermore, global loss of in mice recapitulates maternal and fetal phenotypes of placental insufficiency. dams have reduced spiral artery numbers and late gestational hypertension with resolution following delivery. fetuses are growth restricted and demonstrate changes in umbilical artery Doppler consistent with poor placental perfusion and fetal distress. Loss of increases fetal vascular density in the placenta and dysregulates trophoblast expression of angiogenic factors. Our data support a critical regulatory role for in trophoblast invasion-a necessary process for placentation-representing a possible future target for improving placentation and fetal outcomes.
To better understand the roles of microRNAs in glial function, we used a conditional deletion of Dicer1 (Dicer-CKO) in retinal Müller glia (MG). Dicer1 deletion from the MG leads to an abnormal migration of the cells as early as 1 month after the deletion. By 6 months after Dicer1 deletion, the MG form large aggregations and severely disrupt normal retinal architecture and function. The most highly upregulated gene in the Dicer-CKO MG is the proteoglycan Brevican (Bcan) and overexpression of Bcan results in similar aggregations of the MG in wild-type retina. One potential microRNA that regulates Bcan is miR-9, and overexpression of miR-9 can partly rescue the effects of Dicer1 deletion on the MG phenotype. We also find that MG from retinitis pigmentosa patients display an increase in Brevican immunoreactivity at sites of MG aggregation, linking the retinal remodeling that occurs in chronic disease with microRNAs.
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.
Cell migration in a three-dimensional matrix requires that cells either remodel the surrounding matrix fibers and/or squeeze between the fibers to move. Matrix degradation, matrix remodeling, and changes in cell shape each require cells to expend energy. While significant research has been performed to understand the cellular and molecular mechanisms guiding metastatic migration, less is known about cellular energy regulation and utilization during three-dimensional cancer cell migration. Here we introduce the use of the genetically encoded fluorescent biomarkers, PercevalHR and pHRed, to quantitatively assess ATP, ADP, and pH levels in MDA-MB-231 metastatic cancer cells as a function of the local collagen microenvironment. We find that the use of the probe is an effective tool for exploring the thermodynamics of cancer cell migration and invasion. Specifically, we find that the ATP:ADP ratio increases in cells in denser matrices, where migration is impaired, and it decreases in cells in aligned collagen matrices, where migration is facilitated. When migration is pharmacologically inhibited, the ATP:ADP ratio decreases. Together, our data indicate that matrix architecture alters cellular energetics and that intracellular ATP:ADP ratio is related to the ability of cancer cells to effectively migrate.
© 2018 Zanotelli, Goldblatt, Miller, 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).
Contractile actomyosin bundles, stress fibers, are crucial for adhesion, morphogenesis, and mechanosensing in nonmuscle cells. However, the mechanisms by which nonmuscle myosin II (NM-II) is recruited to those structures and assembled into functional bipolar filaments have remained elusive. We report that UNC-45a is a dynamic component of actin stress fibers and functions as a myosin chaperone in vivo. UNC-45a knockout cells display severe defects in stress fiber assembly and consequent abnormalities in cell morphogenesis, polarity, and migration. Experiments combining structured-illumination microscopy, gradient centrifugation, and proteasome inhibition approaches revealed that a large fraction of NM-II and myosin-1c molecules fail to fold in the absence of UNC-45a. The remaining properly folded NM-II molecules display defects in forming functional bipolar filaments. The C-terminal UNC-45/Cro1/She4p domain of UNC-45a is critical for NM-II folding, whereas the N-terminal tetratricopeptide repeat domain contributes to the assembly of functional stress fibers. Thus, UNC-45a promotes generation of contractile actomyosin bundles through synchronized NM-II folding and filament-assembly activities.
© 2017 Lehtimäki et al.