Bioactive peptides in the juxtamembrane regions of proteins are involved in many signaling events. The juxtamembrane regions of cadherins were examined for the identification of bioactive regions. Several peptides spanning the cytoplasmic juxtamembrane regions of E- and N-cadherin were synthesized and assessed for the ability to influence TGFβ responses in epithelial cells at the gene expression and protein levels. Peptides from regions closer to the membrane appeared more potent inhibitors of TGFβ signaling, blocking Smad3 phosphorylation. Thus inhibiting nuclear translocation of phosphorylated Smad complexes and subsequent transcriptional activation of TGFβ signal propagating genes. The peptides demonstrated a peptide-specific potential to inhibit other TGFβ superfamily members, such as BMP4.
BMP4; E-cadherin; Jagged; N-cadherin; Smad; TGFβ; adherens junctions; palmitic acid; peptide
Highly invasive pancreatic tumors are often recognized in late stages due to a lack of clear symptoms and pose major challenges for treatment and disease management. Broad-band Protein Kinase D (PKD) inhibitors have recently been proposed as additional treatment option for this disease. PKDs are implicated in the control of cancer cell motility, angiogenesis, proliferation and metastasis. In particular, PKD2 expression is elevated in pancreatic cancer, whereas PKD1 expression is comparably lower. In our recent study we report that both kinases control PDAC cell invasive properties in an isoform-specific, but opposing manner. PKD1 selectively mediates anti-migratory/anti-invasive features by preferential regulation of the actin-regulatory Cofilin-phosphatase Slingshot1L (SSH1L). PKD2, on the other hand enhances invasion and angiogenesis of PDAC cells in 3D-ECM cultures and chorioallantois tumor models by stimulating expression and secretion of matrix-metalloproteinase 7 and 9 (MMP7/9). MMP9 also enhances PKD2-mediated tumor angiogenesis releasing extracellular matrix-bound VEGF-A. We thus suggest high PKD2 expression and loss of PKD1 may be beneficial for tumor cells to enhance their matrix-invading abilities. In our recent study we demonstrate for the first time PKD1 and 2 isoform-selective effects on pancreatic cancer cell invasion, in-vitro and in-vivo, defining isoform-specific regulation of PKDs as a major future issue.
PKD1; PKD2; MMP7; MMP9; invasion; angiogenesis; isoform specificity
Current knowledge understands the mesenchymal cell invasion in a 3D matrix as a combined process of cell-to-matrix adhesion based cell migration and matrix remodeling. Excluding cell invasion stimulated by cytokines and chemokines, the basal cell invasion itself is a complicated process that can be regulated by matrix ligand type, density, geometry, and stiffness, etc. Understanding such a complicated biological process requires delicate dissections into simplified model studies by altering only one or two elements at a time. Past cell motility studies focusing on matrix stiffness have revealed that a stiffer matrix promotes 2D X-Y axis lateral cell motility. Here, we comment on two recent studies that report, instead of stiffer matrix, a softer matrix promotes matrix proteolysis and the formation of invadosome-like protrusions (ILPs) along the 3D Z axis. These studies also reveal that soft matrix precisely regulates such ILPs formation in the stiffness scale range of 0.1 kilopascal in normal cells. In contrast, malignant cells such as cancer cells can form ILPs in response to a much wider range of matrix stiffness. Further, different cancer cells respond to their own favorable range of matrix stiffness to spontaneously form ILPs. Thus, we hereby propose the idea of utilizing the matrix stiffness to precisely regulate ILP formation as a mechanophenotyping tool for cancer metastasis prediction and pathological diagnosis.
cell migration; cell invasion; cancer metastasis; invadosomes; invadopodia; podosomes; matrix stiffness; mechanophenotyping; cellular architecture; soft matrix
Age related loss of skeletal muscle mass and function (sarcopenia) reduces independence and the quality of life for individuals, and leads to falls and fractures with escalating health costs for the rapidly aging human population. Thus there is much interest in developing interventions to reduce sarcopenia. One area that has attracted recent attention is the proposed use of myogenic stem cells to improve regeneration of old muscles. This mini-review challenges the fundamental need for myogenic stem cell therapy for sarcopenia. It presents evidence that demonstrates the excellent capacity of myogenic stem cells from very old rodent and human muscles to form new muscles after experimental myofiber necrosis. The many factors required for successful muscle regeneration are considered with a strong focus on integration of components of old muscle bioarchitecture. The fundamental role of satellite cells in homeostasis of normal aging muscles and the incidence of endogenous regeneration in old muscles is questioned. These issues, combined with problems for clinical myogenic stem cell therapies for severe muscle diseases, raise fundamental concerns about the justification for myogenic stem cell therapy for sarcopenia.
sarcopenia; aging skeletal muscle; cell therapy; myogenic stem cells; regeneration
Three different genes each located on a different chromosome encode the heavy chains of nonmuscle myosin II in humans and mice. This review explores the functional consequences of the presence of three isoforms during embryonic development and beyond. The roles of the various isoforms in cell division, cell-cell adhesion, blood vessel formation and neuronal cell migration are addressed in animal models and at the cellular level. Particular emphasis is placed on the role of nonmuscle myosin II during cardiac and brain development, and during closure of the neural tube and body wall. Questions addressed include the consequences on organ development, of lowering or ablating a particular isoform as well as the effect of substituting one isoform for another, all in vivo. Finally the roles of the three isoforms in human diseases such as cancer as well as in syndromes affecting a variety of organs in humans are reviewed.
Cancer; MYH9-RD; Pentalogy of Cantrell; Congenital Diaphragmatic Hernia; Double Outlet of Right Ventricle; Neuronal Migration; Cell Adhesion; Body Wall Closure; Cytokinesis; Karyokinesis; Placental Vascular Formation
Cell surface expansion is a necessary part of cell shape change. One long-standing hypothesis proposes that membrane for this expansion comes from the flattening out of cell surface projections such as microvilli and membrane folds. Correlative EM data of cells undergoing phagocytosis, cytokinesis, and morphogenesis has hinted at the existence of such an unfolding mechanism for decades; but unfolding has only recently been confirmed using live-cell imaging and biophysical approaches. Considering the wide range of cells in which plasma membrane unfolding has now been reported, it likely represents a fundamental mechanism of cell shape change.
cell shape change; microvilli; cell surface area regulation; plasma membrane tension; actin; morphogenesis; exocytosis; cellularization
Columnar epithelia (e.g., kidney, intestine) and hepatocytes embody the two major organizational phenotypes of non-stratified epithelial cells. Columnar epithelia establish their apical and basal domains at opposing poles and organize in monolayered cysts and tubules, in which their apical surfaces form a single continuous lumen whereas hepatocytes establish their apical domains in the midst of their basolateral domains and organize a highly branched capillary luminal network, the bile canaliculi, in which a single hepatocyte can engage in lumen formation with multiple neighbors. To maintain their distinct tissue architectures, columnar epithelial cells bisect their luminal domains during symmetric cell divisions, while the cleavage furrow in dividing hepatocytes avoids bisecting the bile canalicular domains. We discuss recently discovered molecular mechanisms that underlie the different cell division phenotypes in columnar and hepatocytic model cell lines. The serine/threonine kinase Par1b determines both the epithelial lumen polarity and cell division phenotype via cell adhesion signaling that converges on the small GTPase RhoA.
hepatocyte polarity; epithelial cells; mitotic spindle orientation; columnar and hepatocyte polarity; Par1b/MARK2; LGN/NuMA; RhoA
Neural proliferation, migration and differentiation require reorganization of the actin cytoskeleton and regulation of vesicle trafficking to provide stability in maintaining cell adhesions, allow for changes in cell shape, and establishing cell polarity. Human disorders involving the actin-binding Filamin A (FLNA) and vesicle trafficking Brefeldin-associated guanine exchange factor 2 (BIG2 is encoded by the ARFGEF2 gene) proteins are implicated in these various developmental processes, resulting in a malformation of cortical development called periventricular heterotopia (nodules along the ventricular lining) and microcephaly (small brain). Here we discuss several recent reports from our laboratory that demonstrate a shared role for both proteins in actin-associated vesicle trafficking, which is required to maintain the expression and stability of cell adhesion and cell cycle associated molecules during cortical development. While changes in FLNA and BIG2 have first been linked to disorders involving the central nervous system, increasing reports suggest they are associated with aberrant development of various other organ systems in the body. These studies suggest that vesicle trafficking defects in FLN-GEF dependent pathways may contribute to a much broader phenotype than previously realized.
actin; filamin; brefeldin inhibited guanine exchange factor 2; RhoGTPase
Microtubules play a central role in many essential cellular processes, including chromosome segregation, intracellular transport, and cell polarity. As these dynamic polymers are crucial components of eukaryotic cellular architecture, we were surprised by our recent discovery that a common human genetic difference leads to variation in microtubule stability in cells from different people. A single nucleotide polymorphism (SNP) near the TUBB6 gene, encoding class V β-tubulin, is associated with the expression level of this protein, which reduces microtubule stability at higher levels of expression. We discuss the novel cellular GWAS (genome-wide association study) platform that led to this discovery of natural, common variation in microtubule stability and the implications this finding may have for human health and disease, including cancer and neurological disorders. Furthermore, our generalizable approach provides a gateway for cell biologists to help interpret the functional consequences of human genetic variation.
microtubule; genome-wide association; TUBB6; genetic variation; pyroptosis; Salmonella; cellular GWAS; lymphoblastoid
Plasma membrane organization is under the control of cytoskeletal networks and endocytic mechanisms, and a growing literature is showing how closely these influences are interconnected. Here, we review how plasma membranes are formed around individual nuclei of the syncytial Drosophila embryo. Specifically, we outline the pathways that promote and maintain the growth of pseudocleavage and cellularization furrows, as well as specific pathways that keep furrow growth in check. This system has become important for studies of actin regulators, such as Rho1, Diaphanous, non-muscle myosin II and Arp2/3, and endocytic regulators, such as a cytohesin Arf-GEF (Steppke), clathrin, Amphiphysin and dynamin. More generally, it provides a model for understanding how cytoskeletal-endocytic cross-talk regulates the assembly of a cell.
cytoskeleton; endocytosis; plasma membrane; actin; Rho1; cytohesin; Steppke; Amphiphysin; Drosophila; cellularization
Self-organization of dynamic microtubules via interactions with associated motors plays a critical role in spindle formation. The microtubule-based mechanisms underlying other aspects of cellular morphogenesis, such as the formation and development of protrusions from neuronal cells is less well understood. In a recent study, we investigated the molecular mechanism that underlies the massive reorganization of microtubules induced in non-neuronal cells by expression of the neuronal microtubule stabilizer MAP2c. In that study we directly observed cortical dynein complexes and how they affect the dynamic behavior of motile microtubules in living cells. We found that stationary dynein complexes transiently associate with motile microtubules near the cell cortex and that their rapid turnover facilitates efficient microtubule transport. Here, we discuss our findings in the larger context of cellular morphogenesis with specific focus on self-organizing principles from which cellular shape patterns such as the thin protrusions of neurons can emerge.
self-organization; cortical dynein; cytoplasmic dynein; cellular morphogenesis; neuromorphogenesis; neurite; microtubules
Microtubules (MTs) are key cytoskeletal elements in developing and mature neurons. MT reorganization underlies the morphological changes that occur during neuronal development. Furthermore, MTs contribute to the maintenance of neuronal architecture, enable intracellular transport and act as scaffolds for signaling molecules. Thus, a fine-tuned regulation of MT dynamics and stability is crucial for the correct differentiation and functioning of neurons. Different types of proteins contribute to the regulation of the MT state, such as plus-end tracking proteins (+TIPs), which interact with the plus-ends of growing microtubules, and classical microtubule-associated proteins (MAPs), which bind along the microtubule lattice. Recent evidence indicates that MAPs interplay with End Binding Proteins (EBs), the core +TIPs, in neuronal cells. This might contribute to the orchestrated regulation of MT dynamics in neurons. In this mini-review article, we address recent research on the neuronal crosstalk between EBs and classical MAPs and speculate on its possible functional relevance.
microtubule dynamics; neuronal development; MAPs; MAP1B; MAP2; +TIPs; EB proteins
Development of multicellular organisms depends on patterning and growth mechanisms encoded in the genome, but also on the physical properties and mechanical interactions of the constituent cells that interpret these genetic cues. This fundamental biological problem requires integrated studies at multiple levels of biological organization: from genes, to cell behaviors, to tissue morphogenesis. We have recently combined functional genetics with live imaging approaches in embryos of the insect Tribolium castaneum, in order to understand their remarkable transformation from a uniform single-layered blastoderm into a condensed multi-layered embryo covered by extensive extra-embryonic tissues. We first developed a quick and reliable methodology to fluorescently label various cell components in entire Tribolium embryos. Live imaging of labeled embryos at single cell resolution provided detailed descriptions of cell behaviors and tissue movements during normal embryogenesis. We then compared cell and tissue dynamics between wild-type and genetically perturbed embryos that exhibited altered relative proportions of constituent tissues. This systematic comparison led to a qualitative model of the molecular, cellular and tissue interactions that orchestrate the observed epithelial rearrangements. We expect this work to establish the Tribolium embryo as a powerful and attractive model system for biologists and biophysicists interested in the molecular, cellular and mechanical control of tissue morphogenesis.
Tribolium castaneum; Drosophila melanogaster; arthropods; fluorescence live imaging; functional genetics; embryo morphogenesis; extraembryonic development; cell shape; cell intercalation; convergence and extension
Stabilization of axonal connections is an underappreciated, but critical, element in development and maintenance of neuronal functions. The ability to maintain the overall architecture of the brain for decades is essential for our ability to process sensory information efficiently, coordinate motor activity, and retain memories for a lifetime. While the importance of the neuronal cytoskeleton in this process is acknowledged, little has been known about specializations of the axonal cytoskeleton needed to stabilize neuronal architectures. A novel post-translational modification of tubulin that stabilizes normally dynamic microtubules in axons has now been identified. Polyamination appears to be enriched in axons and is developmentally regulated with a time course that correlates with increased microtubule stabilization. Identifying one of the molecular mechanisms for maintaining neuronal connections creates new research avenues for understanding the role of stabilizing neuronal architecture in neuronal function and in neuropathology.
axonal cytoskeleton; microtubule stability; plasticity; post-translational modification; neuronal function
Cadherin-mediated cell adhesion at Adherens Junctions (AJs) and its dynamic connections with the microtubule (MT) cytoskeleton are important regulators of cellular architecture. However, the functional relevance of these interactions and the molecular players involved in different cellular contexts and cellular compartments are still not completely understood. Here, we comment on our recent findings showing that the MT plus-end binding protein CLASP2 interacts with the AJ component p120-catenin (p120) specifically in progenitor epidermal cells. Absence of either protein leads to alterations in MT dynamics and AJ functionality. These findings represent a novel mechanism of MT targeting to AJs that may be relevant for the maintenance of proper epidermal progenitor cell homeostasis. We also discuss the potential implication of other MT binding proteins previously associated to AJs in the wider context of epithelial tissues. We hypothesize the existence of adaptation mechanisms that regulate the formation and stability of AJs in different cellular contexts to allow the dynamic behavior of these complexes during tissue homeostasis and remodeling.
microtubules; Adherens Junctions; p120-catenin; CLASP2; cadherins; epidermis; keratinocytes
Epiboly, the thinning and spreading of one tissue over another, is a widely employed morphogenetic movement that is essential for the development of many organisms. In the zebrafish embryo, epiboly describes the coordinated vegetal movement of the deep cells, enveloping layer (EVL) and yolk syncytial layer (YSL) to engulf the yolk cell. Recently, we showed that the large GTPase Dynamin plays a fundamental role in epiboly in the early zebrafish embryo. Because Dynamin plays a well-described role in vesicle scission during endocytosis, we predicted that Dynamin might regulate epiboly through participating in bulk removal of the yolk cell membrane ahead of the advancing margin, a proposed part of the epiboly motor. Unexpectedly, we found that Dynamin function was dispensable in the yolk cell and instead, it was required to maintain the epithelial integrity of the EVL during epiboly. Here, we present a model describing the maintenance of EVL integrity, which is required for the proper generation and transmission of tension during epiboly. Furthermore, we discuss the role of Dynamin-mediated regulation of ezrin-radixin-moesin (ERM) family proteins in the maintenance of epithelial integrity.
zebrafish; epiboly; dynamin; enveloping layer; endocytosis; epithelial integrity; ERM proteins; synaptotagmin
Recent studies have revealed a novel mechanism of myosin regulation in which the actin-binding protein tropomyosin converts atypical type-V myosins into processive cargo transporters. To achieve this, tropomyosin's primary role appears to lie in its ability to influence myosin's enzyme kinetics, prolonging the strong actin-bound ADP/apo state to enable hand-over-hand walking of myosin-V dimers along actin tracks. Activation of myosin-V mediated transport by tropomyosin underscores its function in helping to direct cargos to specific actin tracks and subcellular destinations. This type of regulation supports the broader notion that tropomyosin plays a key role in actomyosin sorting.
actin; tropomyosin; myosin-V; duty ratio; processivity; intracellular transport; actomyosin sorting
Clathrin-independent endocytosis (CIE) mediates the internalization of many plasma membrane (PM) proteins involved in homeostasis, immune response, and signaling. CIE cargo molecules are internalized independent of clathrin, and dynamin, and modulated by the small G protein Arf6. After internalization the CIE cargo proteins either follow a default pathway of trafficking to lysosomes for degradation or follow a pathway where they are routed directly to the recycling endosomes for return to the PM. The selective endosomal sorting of molecules like CD44, CD98, and CD147, which are involved in cell-cell and cell-extracellular interactions, indicates that sorting mechanisms dictate the post-endocytic fate of CIE cargo proteins. In a recent study, we identified sorting signals that specify the endosomal trafficking of CIE cargo proteins and uncover a role for Hook1 as an endosomal cargo adaptor that routes CIE cargo to the recycling endosomes. Furthermore, we found that Hook1, microtubules, and Rab22a work in coordination to directly recycle the cargo and facilitate cell spreading. Here, we discuss our current view on the endosomal sorting of CIE cargo proteins and their molecular regulators.
clathrin-independent endocytosis; Hook1; Rab22a; Rab22; microtubules; endosomal sorting; sorting signals; recycling; basigin; CD147
Streptomyces is a multicellular mycelial bacterium, which exhibits pronounced cell polarity and grows by extension of the hyphal tips. Similarly to other polarly growing walled cells, such as filamentous fungi or pollen tubes, Streptomyces hyphae face an intrinsic problem: addition of new cell wall material causes structural weakness of the elongating tip. Cellular strategies employed by walled cells to cope with this problem are not well understood. We have identified a coiled coil protein FilP, with properties similar to those of animal intermediate filament (IF) proteins, which somehow confers rigidity and elasticity to the Streptomyces hyphae. In a recent publication we showed that FilP forms extensive cis-interconnected networks, which likely explain its biological function in determining the mechanical properties of the cells. Surprisingly, the intrinsically non-dynamic cytoskeletal network of FilP exhibits a dynamic behavior in vivo and assembles into growth-dependent polar gradients. We show that apical accumulation of FilP is dependent on its interaction with the main component of the Streptomyces polarisome, DivIVA. Thus, the same polarisome complex that orchestrates cell elongation, also recruits an additional stress-bearing structure to the growing tips with an intrinsically weak cell wall. Similar strategy might be used by all polarly growing walled cells.
intermediate filament; cytoskeleton; polar growth; Streptomyces; coiled coil; FilP
The cytoskeleton is a dynamic three dimensional structure contained within the cytoplasm of a cell, and is important in cell shape and movement, and in metastatic progression during carcinogenesis. Members of the Rho family of small GTPases, RHO, RAC and cell cycle division 42 (Cdc42) proteins regulate cytoskeletal dynamics, through the control of a panel of genes. We have recently shown that the microRNA (miRNA) miR-23b represents a central effector of cytoskeletal remodelling. It increases cell-cell interactions, modulates focal adhesion and reduces cell motility and invasion by directly regulating several genes involved in these processes.
miR-23b; microRNAs; miRNAs; cytoskeleton; motility; metastasis; adhesion; focal adhesion; tumor growth
Determination of primordial germ cells (PGCs) is one of the earliest decisions in animal embryogenesis. In many species, PGCs are determined through maternally-inherited germ plasm ribonucleoparticles (RNPs). In zebrafish, these are transmitted during oogenesis as dispersed RNPs, which after fertilization multimerize and become recruited as large aggregates at furrows for the first and second cell cycles. Here, we show that the number of recruited germ plasm RNPs is halved every cell cycle. We also show that germ plasm RNPs are recruited during the third cell cycle, but only transiently. Our data support a mechanism in which systematic local gathering of germ plasm RNPs during cytokinesis and threshold-dependent clearing contribute to forming germ plasm aggregates with the highest RNP number and germ cell-inducing potential.
zebrafish; germ plasm; germ cell; ribonucleoparticles; maternal inheritance; cytokinesis; chromosomal passenger complex; Birc5b; microtubule asters
Nonmuscle myosin-II is an actin-based motor that converts chemical energy into force and movement, and thus functions as a key regulator of the eukaryotic cytoskeleton. Although it is established that phosphorylation on the regulatory light chain increases the actin-activated MgATPase activity of the motor and promotes myosin-II filament assembly, studies have begun to characterize alternative mechanisms that regulate filament assembly and disassembly. These investigations have revealed that all three nonmuscle myosin-II isoforms are subject to additional regulatory controls, which impact diverse cellular processes. In this review, we discuss current knowledge on mechanisms that regulate the oligomerization state of nonmuscle myosin-II filaments by targeting the myosin heavy chain.
nonmuscle myosin-II; NMHC-IIA; NMHC-IIB; NMHC-IIC; phosphorylation; S100A4; Lgl; filament assembly
Neurons begin their life as simple spheres, but can ultimately assume an elaborate morphology with numerous, highly arborized dendrites, and long axons. This is achieved via an astounding developmental progression which is dependent upon regulated assembly and dynamics of the cellular cytoskeleton. As neurites emerge out of the soma, neurons break their spherical symmetry and begin to acquire the morphological features that define their structure and function. Neurons regulate their cytoskeleton to achieve changes in cell shape, velocity, and direction as they migrate, extend neurites, and polarize. Of particular importance, the organization and dynamics of actin and microtubules directs the migration and morphogenesis of neurons. This review focuses on the regulation of intrinsic properties of the actin and microtubule cytoskeletons and how specific cytoskeletal structures and dynamics are associated with the earliest phase of neuronal morphogenesis—neuritogenesis.
neuronal morphogenesis; neurite formation; cytoskeleton; actin; microtubules
Tropomyosin is an actin binding protein that regulates actin filament dynamics and its interactions with actin binding proteins such as myosin, tropomodulin, formin, Arp2/3 and ADF-cofilin in most eukaryotic cells. Tropomyosin is the prototypical two-chained, α-helical coiled coil protein that associates end-to-end and binds to both sides of the actin filament. Each tropomyosin molecule spans four to seven actin monomers in the filament, depending on the size of the tropomyosin. Tropomyosins have a periodic heptad repeat sequence that is characteristic of coiled coil proteins as well as additional periodicities required for its interaction with the actin filament, where each periodic repeat interacts with one actin molecule. This review addresses the role of periodic features of the Tm molecule in carrying out its universal functions of binding to the actin filament and its regulation and the specific features that may determine the isoform specificity of tropomyosins.
tropomyosin; muscle regulation; actin filament; cytoskeleton; coiled coil
The notochord is an evolutionarily conserved structure that has long been known to play an important role in patterning during embryogenesis. Structurally, the notochord is composed of two cell layers: an outer epithelial-like sheath, and an inner core of cells that contain large fluid-filled vacuoles. We have recently shown these notochord vacuoles are lysosome-related organelles that form through Rab32a and vacuolar-type proton-ATPase-dependent acidification. Disruption of notochord vacuoles results in a shortened embryo along the anterior-posterior axis. Interestingly, we discovered that notochord vacuoles are also essential for proper spine morphogenesis and that vacuole defects lead to scoliosis of the spine. Here we discuss the cellular organization of the notochord and how key features of its architecture allow the notochord to function in embryonic axis elongation and spine formation.
zebrafish; notochord vacuole; lysosome-related organelle; axis elongation; spine formation; scoliosis