Peripheral Protrusions Research Articles

A KIF1C-CNBP motor-adaptor complex for trafficking mRNAs to cell protrusions.

mRNA localization to subcellular compartments is a widely used mechanism that functionally contributes to numerous processes. mRNA targeting can be achieved upon recognition of RNA cargo by molecular motors. However, our molecular understanding of how this is accomplished is limited, especially in higher organisms. We focus on a pathway that targets mRNAs to peripheral protrusions of mammalian cells and is important for cell migration. Trafficking occurs through active transport on microtubules, mediated by the KIF1C kinesin. Here, we identify the RNA-binding protein CNBP, as a factor required for mRNA localization to protrusions. CNBP binds directly to GA-rich sequences in the 3'UTR of protrusion targeted mRNAs. CNBP also interacts with KIF1C and is required for KIF1C recruitment to mRNAs and for their trafficking on microtubules to the periphery. This work provides a molecular mechanism for KIF1C recruitment to mRNA cargo and reveals a motor-adaptor complex for mRNA transport to cell protrusions.

A multiscale whole-cell theory for mechanosensitive migration on viscoelastic substrates

Increasing experimental evidence validates that both the elastic stiffness and viscosity of the extracellular matrix regulate mesenchymal cell behavior, such as the rational switch between durotaxis (cell migration to stiffer regions), anti-durotaxis (migration to softer regions), and adurotaxis (stiffness-insensitive migration). To reveal the mechanisms underlying the crossover between these motility regimes, we have developed a multiscale chemomechanical whole-cell theory for mesenchymal migration. Our framework couples the subcellular focal adhesion dynamics at the cell-substrate interface with the cellular cytoskeletal mechanics and the chemical signaling pathways involving Rho GTPase proteins. Upon polarization by the Rho GTPase gradients, our simulated cell migrates by concerted peripheral protrusions and contractions, a hallmark of the mesenchymal mode. The resulting cell dynamics quantitatively reproduces the experimental migration speed as a function of the uniform substrate stiffness and explains the influence of viscosity on the migration efficiency. In the presence of stiffness gradients and absence of chemical polarization, our simulated cell can exhibit durotaxis, anti-durotaxis, and adurotaxis respectively with increasing substrate stiffness or viscosity. The cell moves toward an optimally stiff region from softer regions during durotaxis and from stiffer regions during anti-durotaxis. We show that cell polarization through steep Rho GTPase gradients can reverse the migration direction dictated by the mechanical cues. Overall, our theory demonstrates that opposing durotactic behaviors emerge via the interplay between intracellular signaling and cell-medium mechanical interactions in agreement with experiments, thereby elucidating complex mechanosensing at the single-cell level.

A Novel MEMS Capacitive Microphone with Semiconstrained Diaphragm Supported with Center and Peripheral Backplate Protrusions.

Audio applications such as mobile phones, hearing aids, true wireless stereo earphones, and Internet of Things devices demand small size, high performance, and reduced cost. Microelectromechanical system (MEMS) capacitive microphones fulfill these requirements with improved reliability and specifications related to sensitivity, signal-to-noise ratio (SNR), distortion, and dynamic range when compared to their electret condenser microphone counterparts. We present the design and modeling of a semiconstrained polysilicon diaphragm with flexible springs that are simply supported under bias voltage with a center and eight peripheral protrusions extending from the backplate. The flexible springs attached to the diaphragm reduce the residual film stress effect more effectively compared to constrained diaphragms. The center and peripheral protrusions from the backplate further increase the effective area, linearity, and sensitivity of the diaphragm when the diaphragm engages with these protrusions under an applied bias voltage. Finite element modeling approaches have been implemented to estimate deflection, compliance, and resonance. We report an 85% increase in the effective area of the diaphragm in this configuration with respect to a constrained diaphragm and a 48% increase with respect to a simply supported diaphragm without the center protrusion. Under the applied bias, the effective area further increases by an additional 15% as compared to the unbiased diaphragm effective area. A lumped element model has been also developed to predict the mechanical and electrical behavior of the microphone. With an applied bias, the microphone has a sensitivity of −38 dB (ref. 1 V/Pa at 1 kHz) and an SNR of 67 dBA measured in a 3.25 mm × 1.9 mm × 0.9 mm package including an analog ASIC.

RNA localization and co‐translational interactions control RAB13 GTPase function and cell migration

Numerous RNAs exhibit specific distribution patterns in mammalian cells. However, the functional and mechanistic consequences are relatively unknown. Here, we investigate the functional role of RNA localization at cellular protrusions of migrating mesenchymal cells, using as a model the RAB13 RNA, which encodes a GTPase important for vesicle‐mediated membrane trafficking. While RAB13 RNA is enriched at peripheral protrusions, the expressed protein is concentrated perinuclearly. By specifically preventing RAB13 RNA localization, we show that peripheral RAB13 translation is not important for the overall distribution of the RAB13 protein or its ability to associate with membranes, but is required for full activation of the GTPase and for efficient cell migration. RAB13 translation leads to a co‐translational association of nascent RAB13 with the exchange factor RABIF. Our results indicate that RAB13‐RABIF association at the periphery is required for directing RAB13 GTPase activity to promote cell migration. Thus, translation of RAB13 in specific subcellular environments imparts the protein with distinct properties and highlights a means of controlling protein function through local RNA translation.

Thermal analysis and experimental validation of pin fins with peripheral protrusions

With the growing pace of technological advancement, electronic and thermal components play a crucial role in every domain. These components are however challenged by continuous heat generation which is required to be dissipated for their efficient working. This gives rise to the need of heat dissipating measures and hence, an extensive research is being done in this field. Extended surfaces, known as fins provide an efficient way of cooling components and their utility ranges from medical, power, thermal to microelectronic components. The present work is aimed to augment the thermal performance of pin fins by the introduction of protrusions on the surface of the fin along the length, in two rows, each row having three protrusions. Different geometrical configurations viz. cuboidal, cylindrical, triangular and hemispherical for the protrusion were analysed using Computational Fluid Dynamics. From the results, it is evident that the maximum increase of approximately 7% of the total heat flux is obtained from the surface of the fin with cylindrical protrusions and considerable increase in the total heat flux is obtained in other configurations. It was also concluded that cuboidal and cylindrical protrusions have lowest temperature profiles indicating better heat transfer behaviour. The temperature profile for the fin without protrusions was experimentally validated. The results of the fins with protrusions are in agreement with the existing research papers.

The effect of Rho drugs on mast cell activation and degranulation.

Mast cells are tissue-resident immune cells that produce potent proinflammatory mediators, which are stored in cytoplasmic granules. Stimulation triggers degranulation, a process that mobilizes granules to dock and fuse to the plasma membrane, releasing mediators. Mast cell degranulation has an important role in immunity but can also intensify inflammation and contribute to allergic disorders. Hence, it is important to understand signaling pathways that regulate mast cell degranulation. Here, we examined the role of Rho proteins in regulating mast cell activation leading to degranulation. RBL-2H3 cells and bone marrow-derived mast cells (BMMCs) were stimulated through aggregation of FcεRI receptors. Stimulated cells showed a large increase in the levels of activated Rac and, to a lesser extent, RhoA. Drugs were used to acutely inhibit the function of specific Rho proteins. The Rac inhibitor EHT-1864 and the RhoA inhibitor rhosin inhibited degranulation. Microscopic characterization showed that, upon stimulation, RBL-2H3 cells formed surface ridges that grew into large protrusions reminiscent of circular dorsal ruffles, which flattened into large lamellipodia. LysoTracker-labeled cells showed granules stream into peripheral protrusions. EHT-1864 reduced granule motility, whereas rhosin increased motility; both drugs affected the formation of peripheral protrusions. These results showed that, in response to stimuli, Rho proteins control discrete cytoskeletal remodeling processes that are needed for granule exocytosis. Rac is required to stimulate the remodeling of mast cells, triggering actin-mediated flattening of the cell periphery to create an active degranulation zone, whereas RhoA controls the streaming of highly motile granules into the active zone.

The Dual Organization of P-bodies Revealed by Immunoelectron Microscopy and Electron Tomography

Processing bodies (P-bodies) are cytoplasmic non-membranous domains involved in the regulation of eukaryotic gene expression. Since their discovery, several studies using fluorescence-based strategies have uncovered their pivotal role in mRNA metabolism, particularly during translation repression and/or mRNA degradation. Yet, P-bodies still remain a “black box” in which numerous proteins accumulate next to RNAs to regulate their fate by unknown mechanisms. In this study, we investigated the ultrastructural organization of P-bodies in human cells. Using a wide range of original electron microscopy strategies, including high-pressure freezing and freeze substitution, we found that P-bodies are huge ribonucleoprotein complexes located in the close proximity of mitochondria and ribosomes, in which regulatory factors exhibit differential localization depending on their activity on mRNAs. We describe the first experiment pairing immunogold labeling with electron tomography (immunoelectron tomography) of a human P-body. Overall, the results depict a P-body organization that comprises at least two distinct compartments: a dense core on which peripheral protrusions are anchored.

Cryo-Electron Microscopy Structure of a Yeast Mitochondrial Preprotein Translocase

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for proteins imported into mitochondria. We determined the structure of the native, unstained ∼ 550-kDa core–Tom20 complex from Saccharomyces cerevisiae by cryo-electron microscopy at 18-Å resolution. The complex is triangular, measuring 145 Å on edge, and has near-3-fold symmetry. Its bulk is made up of three globular ∼ 50-Å domains. Three elliptical pores on the c-face merge into one central ∼ 70-Å cavity with a cage-like assembly on the opposite t-face. Nitrilotriacetic acid–gold labeling indicates that three Tom22 subunits in the TOM complex are located at the perimeter of the complex near the interface of the globular domains. We assign Tom22, which controls complex assembly, to three peripheral protrusions on the c-face, while the Tom20 subunit is tentatively assigned to the central protrusion on this surface. Based on our three-dimensional map, we propose a model of transient interactions and functional dynamics of the TOM assembly.

The Effects of Colloidal Nanotopography on Initial Fibroblast Adhesion and Morphology

Colloidal lithography offers a simple, inexpensive method of producing irregular nanotopographies, a pattern not easily attainable utilizing conventional serial writing processes. Colloids with 20- or 50-nm diameter were utilized to produce such an irregular topography and were characterized by calculating the percentage area coverage of particles. Interparticle and nearest neighbor spacing were also assessed for the individual colloids in the pattern. Two-way analysis of variance (ANOVA) indicated significant differences between the number of fibroblasts adhering to planar, 20-, and 50-nm-diameter colloidal topographies, the number of fibroblasts adhering to the substrates at the time intervals studied, namely 20 min, 1 h, and 3 h and significant interaction between time and topography on fibroblast adhesion (P < 0.01). Tukey tests were utilized for sensitive identification of the differences between the sample means and compounded ANOVA results. Cytoskeletal and general cell morphology were investigated on planar and colloidal substrates, and indicated cells in contact with irregular nanotopographies exhibit many peripheral protrusions while such protrusions are absent in cells on planar control surfaces. These protrusions are rich in microtubules on 20-nm-diameter colloidal surfaces while microfilaments are prevalent on 50-nm-diameter surfaces. Moreover, by 3 h, cells on the colloidal substrates initiate cell-cell adhesions, also absent in controls.

The WASP-Binding Protein WIRE Has a Role in the Regulation of the Actin Filament System Downstream of the Platelet-Derived Growth Factor Receptor

Activation of growth factor receptors, such as platelet-derived growth factor (PDGF) receptors, has a major impact on the motile behavior of vertebrate cells. The WASP family of proteins has been recognized as important regulators of actin polymerization via the activation of the Arp2/3 complex. The activity of the WASP proteins has, in turn, been shown to be governed by a number of associated proteins, including the WASP interacting protein (WIP). This report presents a novel WIP-like protein, WIRE (for WI P- re lated). WIRE was shown to bind to the WH1 domain of WASP and N-WASP. WIRE was localized to actin filaments in transiently transfected PAE/PDGFRβ cells, and in cells simultaneously expressing WIRE and WASP, WIRE relocalized WASP to actin filaments, a relocalization that required direct interaction between the two proteins. In addition, WIRE was able to bind the PDGF receptor substrate Nckβ. PDGF treatment of cells ectopically expressing WIRE resulted in formation of peripheral protrusions composed of filopodia and lamellipodia-like structures. In cells expressing both WIRE and WASP, PDGF treatment induced a translocation of WASP to the cell margin, an effect that required the presence of WIRE. Taken together, the data presented indicate that WIRE has a role in the WASP-mediated organization of the actin cytoskeleton and that WIRE is a potential link between the activated PDGF receptor and the actin polymerization machinery.

Morphology of water-insoluble and -soluble glucans of the dental plaque organism Streptococcus mutans AHT and their interaction with the cell surface envelope

Glucans synthesized from sucrose by cell-free glucosyltransferase of Streptococcus mutans AHT were separated into water-insoluble (ISG) and water-soluble (SG) fractions by centrifugation. The isolated ISG comprised coalescent clumps of two fibrillar components. One component consisted of two protofibrils approx. 4nm thick in a parallel arrangement and bearing short fluffy protrusions on the side surface. The other component of ISG, and the isolated SG also, was a single-stranded fibril, about 2 nm thick, usually in fine meshworks and sometimes thick clumps. Dextranase treatment of ISG removed the single-stranded fibrils and the fluffy peripheral protrusions of the double strands. The results indicate that ISG is a mixture of fibrillar components, and suggest that glucans produced by Strep. mutans AHT can be classified into two different types: an inherently water-insoluble, dextranase-insensitive, double-stranded fibril having dextranase-sensitive peripheral fluffy protrusions, and a separate dextranase-sensitive single-stranded fibril, present free in aqueous solution or in coherent association with water-insoluble double strands. The double strands of ISG were associated tangentially with the fuzzy coat of Strep. mutans AHT cells via their peripheral protrusions, whereas the single strands of ISG and SG attached perpendicularly. The residual-core double strands of dextranase-treated ISG did not attach to the fuzzy coat. ISG and SG did not bind to heat-treated cells, which showed no detectable morphological alteration of the cell surface envelope, nor did they bind to trypsin-treated cells lacking the bulk of the fuzzy coat. Recultivated in glucose broth, trypsinized cells recovered glucan-binding activity before detectable reconstruction of the fuzzy coat, to which the two types of fibrillar glucans attached in their characteristic manners, thus confirming that α-l,6-linked glucose residues of ISG and SG and a heat-labile, proteinaceous component of the cell-surface fuzzy coat participate in the glucan-induced agglutination of Strep. mutans cells.

Possible formation and development of spirochaete attachment sites found on the surface of symbiotic polymastigote flagellates of the termite Reticulitermes flavipes

We propose that spirochaete attachment sites arise from peripheral protrusions which appear on the surface of polymastigote flagellates. These protrusions develop into bracket-shaped structures which then form the mature attachment site. Next the site becomes detached from the surface of the cell; this latter process may be facilitated by the fusion of vesicles located in the region immediately beneath the spirochaete attachment site. This theory could explain the variability in the number and distribution of attachment sites on the surface of the flagellates.

Morphology, biochemical analysis and neuraminidase activity of rubella virus.

A simple and reproducible method for the production of purified rubella virus is described. Purified virus was subjected to morphological and chemical analysis. The virus particles were rather pleomorphic (60 nm diameter), sometimes with one or more peripheral protrusions. The viral surface, revealed by negative staining, was composed of spikes 6 nm long, featuring enlarged ends. In SDS-urea-polyacrylamide gel electrophoresis, 4 major and 4 minor polypeptide bands were revealed. Total lipids and phospholipids were analysed on the same preparation. The viral particles were composed of RNA: 0.030 mg, and lipids: 0.245 mg, of which 0.169 mg were phospholipids for each mg of viral protein. Biologically, the purified virus preparation showed high infectivity, a high hemagglutination titre and a weak neuraminidase activity under defined conditions.

The Merkel Cell

The Merkel cell is a distinctive cell normally found in the epidermis of mammals. It possesses a folded nucleus; a clear, organelle-rich cytoplasm with peripheral protrusions among the epithelial cells; and a few desmosomal attachments to adjacent cells. The cytoplasm is characterized by many dense-cored, membrane-bound granucles, 80 to 100 nm in diameter and distributed in the cytoplasm next to the nerve termination. The Merkel cell is found isolated in the epidermis or associated with epithelial nerve endings such as the hederiform ending of Merkel-Ranvier, the tactile hair disk of pinkus, and the organ of Eimer. It may be observed in the dermis in relation to nerve trunks and also within the corpuscles of Grandry found in the skin of the duck bill. The merkel cell appears to be an individual cell type, probably derived from the neural crest. Which is capable of living in union with neural and epithelial cells. The Merkel cell penetrates the epidermis in fetal life and functions as a specific, slowly adapting, sensory touch receptor.