scholarly journals Photoreceptor disc membranes are formed through an Arp2/3-dependent lamellipodium-like mechanism

2019 ◽  
Vol 116 (52) ◽  
pp. 27043-27052 ◽  
Author(s):  
William J. Spencer ◽  
Tylor R. Lewis ◽  
Sebastien Phan ◽  
Martha A. Cady ◽  
Ekaterina O. Serebrovskaya ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Time Course ◽  
Conditional Knockout ◽  
Actin Network ◽  
Actin Networks ◽  
Conditional Knockout Mouse ◽  
Proteomic Approach ◽  
Vertebrate Photoreceptor ◽  
Actin Nucleator ◽  
Disc Formation

The light-sensitive outer segment of the vertebrate photoreceptor is a highly modified primary cilium filled with disc-shaped membranes that provide a vast surface for efficient photon capture. The formation of each disc is initiated by a ciliary membrane evagination driven by an unknown molecular mechanism reportedly requiring actin polymerization. Since a distinct F-actin network resides precisely at the site of disc morphogenesis, we employed a unique proteomic approach to identify components of this network potentially driving disc morphogenesis. The only identified actin nucleator was the Arp2/3 complex, which induces the polymerization of branched actin networks. To investigate the potential involvement of Arp2/3 in the formation of new discs, we generated a conditional knockout mouse lacking its essential ArpC3 subunit in rod photoreceptors. This knockout resulted in the complete loss of the F-actin network specifically at the site of disc morphogenesis, with the time course of ArpC3 depletion correlating with the time course of F-actin loss. Without the actin network at this site, the initiation of new disc formation is completely halted, forcing all newly synthesized membrane material to be delivered to the several nascent discs whose morphogenesis had already been in progress. As a result, these discs undergo uncontrolled expansion instead of normal enclosure, which leads to formation of unusual, large membrane whorls. These data suggest a model of photoreceptor disc morphogenesis in which Arp2/3 initiates disc formation in a “lamellipodium-like” mechanism.

scholarly journals Specificity and Redundancy of Profilin 1 and 2 Function in Brain Development and Neuronal Structure

Cells ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 2310
Author(s):  
Marina Di Domenico ◽  
Melanie Jokwitz ◽  
Walter Witke ◽  
Pietro Pilo Boyl
Keyword(s):  
Actin Polymerization ◽  
Conditional Knockout ◽  
Brain Morphology ◽  
Actin Dynamics ◽  
Neuronal Structure ◽  
Knockout Mouse Model ◽  
Conditional Knockout Mouse ◽  
Neuronal Stem Cell ◽  
Neuronal Precursors ◽  
Redundant Functions

Profilin functions have been discussed in numerous cellular processes, including actin polymerization. One puzzling aspect is the concomitant expression of more than one profilin isoform in most tissues. In neuronal precursors and in neurons, profilin 1 and profilin 2 are co-expressed, but their specific and redundant functions in brain morphogenesis are still unclear. Using a conditional knockout mouse model to inactivate both profilins in the developing CNS, we found that threshold levels of profilin are necessary for the maintenance of the neuronal stem-cell compartment and the generation of the differentiated neurons, irrespective of the specific isoform. During embryonic development, profilin 1 is more abundant than profilin 2; consequently, modulation of profilin 1 levels resulted in a more severe phenotype than depletion of profilin 2. Interestingly, the relevance of the isoforms was reversed in the postnatal brain. Morphology of mature neurons showed a stronger dependence on profilin 2, since this is the predominant isoform in neurons. Our data highlight redundant functions of profilins in neuronal precursor expansion and differentiation, as well as in the maintenance of pyramidal neuron dendritic arborization. The specific profilin isoform is less relevant; however, a threshold profilin level is essential. We propose that the common activity of profilin 1 and profilin 2 in actin dynamics is responsible for the observed compensatory effects.

scholarly journals Reconstitution of contractile actomyosin rings in vesicles

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Thomas Litschel ◽  
Charlotte F. Kelley ◽  
Danielle Holz ◽  
Maral Adeli Koudehi ◽  
Sven K. Vogel ◽  
...  
Keyword(s):  
Large Scale ◽  
Actin Polymerization ◽  
Living Cells ◽  
Actin Ring ◽  
Actin Networks ◽  
Mechanical Transformation ◽  
Myosin Motors ◽  
Spherical Vesicles ◽  
Theoretical Considerations ◽  
Encapsulation Methods

AbstractOne of the grand challenges of bottom-up synthetic biology is the development of minimal machineries for cell division. The mechanical transformation of large-scale compartments, such as Giant Unilamellar Vesicles (GUVs), requires the geometry-specific coordination of active elements, several orders of magnitude larger than the molecular scale. Of all cytoskeletal structures, large-scale actomyosin rings appear to be the most promising cellular elements to accomplish this task. Here, we have adopted advanced encapsulation methods to study bundled actin filaments in GUVs and compare our results with theoretical modeling. By changing few key parameters, actin polymerization can be differentiated to resemble various types of networks in living cells. Importantly, we find membrane binding to be crucial for the robust condensation into a single actin ring in spherical vesicles, as predicted by theoretical considerations. Upon force generation by ATP-driven myosin motors, these ring-like actin structures contract and locally constrict the vesicle, forming furrow-like deformations. On the other hand, cortex-like actin networks are shown to induce and stabilize deformations from spherical shapes.

scholarly journals Irradiation of Nf1 mutant mouse models of spinal plexiform neurofibromas drives pathologic progression and decreases survival

2021 ◽  
Author(s):  
Danny Laurent ◽  
Abbi E Smith ◽  
Waylan K Bessler ◽  
Marc Mendonca ◽  
Helen Chin-Sinex ◽  
...  
Keyword(s):  
Overall Survival ◽  
Mouse Models ◽  
Conditional Knockout ◽  
Benign Neoplasms ◽  
Neoplastic Progression ◽  
Plexiform Neurofibromas ◽  
Normal Tissues ◽  
Peripheral Nerve Sheath Tumors ◽  
Conditional Knockout Mouse ◽  
Therapeutic Irradiation

Abstract Background Genetically susceptible individuals can develop malignancies after irradiation of normal tissues. In the context of therapeutic irradiation, it is not known whether irradiating benign neoplasms in susceptible individuals promotes neoplastic transformation and worse clinical outcomes. Individuals with Neurofibromatosis 1 (NF1) are susceptible to both radiation-induced second malignancies and spontaneous progression of plexiform neurofibromas (PNs) to malignant peripheral nerve sheath tumors (MPNSTs). The role of radiotherapy in the treatment of benign neoplasms such as PNs is unclear. Methods To test whether radiotherapy promotes neoplastic progression of PNs and reduces overall survival, we administered spinal irradiation (SI) to conditional knockout mouse models of NF1-associated PNs in two germline contexts: Nf1 fllfl; PostnCre + and Nf1 fl/-; PostnCre +. Both genotypes develop extensive Nf1 null spinal PNs, modeling PNs in NF1 patients. A total of 101 mice were randomized to 0 Gy, 15 Gy (3 Gy x 5), or 30 Gy (3 Gy x 10) of spine-focused, fractionated SI and aged until signs of illness. Results SI decreased survival in both Nf1 fllfl mice and Nf1 fl/- mice, with the worst overall survival occurring in Nf1 fl/- mice receiving 30 Gy. SI was also associated with increasing worrisome histologic features along the PN-MPNST continuum in PNs irradiated to higher radiation doses. Conclusions This pre-clinical study provides experimental evidence that irradiation of pre-existing PNs reduces survival and may shift PNs to higher grade neoplasms.

Decline of contractility during ischemia-reperfusion injury: actin glutathionylation and its effect on allosteric interaction with tropomyosin

2006 ◽  
Vol 290 (3) ◽  
pp. C719-C727 ◽  
Author(s):  
Frank C. Chen ◽  
Ozgur Ogut
Keyword(s):  
Reperfusion Injury ◽  
Posttranslational Modifications ◽  
Actin Polymerization ◽  
Time Course ◽  
Troponin I ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
High Concentration ◽  
Cross Sectional ◽  
The Impact

The severity and duration of ischemia-reperfusion injury is hypothesized to play an important role in the ability of the heart subsequently to recover contractility. Permeabilized trabeculae were prepared from a rat model of ischemia-reperfusion injury to examine the impact on force generation. Compared with the control perfused condition, the maximum force (Fmax) per cross-sectional area and the rate of tension redevelopment of Ca2+-activated trabeculae fell by 71% and 44%, respectively, during ischemia despite the availability of a high concentration of ATP. The reduction in Fmax with ischemia was accompanied by a decline in fiber stiffness, implying a drop in the absolute number of attached cross bridges. However, the declines during ischemia were largely recovered after reperfusion, leading to the hypothesis that intrinsic, reversible posttranslational modifications to proteins of the contractile filaments occur during ischemia-reperfusion injury. Examination of thin-filament proteins from ischemic or ischemia-reperfused hearts did not reveal proteolysis of troponin I or T. However, actin was found to be glutathionylated with ischemia. Light-scattering experiments demonstrated that glutathionylated G-actin did not polymerize as efficiently as native G-actin. Although tropomyosin accelerated the time course of native and glutathionylated G-actin polymerization, the polymerization of glutathionylated G-actin still lagged native G-actin at all concentrations of tropomyosin tested. Furthermore, cosedimentation experiments demonstrated that tropomyosin bound glutathionylated F-actin with significantly reduced cooperativity. Therefore, glutathionylated actin may be a novel contributor to the diverse set of posttranslational modifications that define the function of the contractile filaments during ischemia-reperfusion injury.

scholarly journals Ras-mediated activation of the TORC2–PKB pathway is critical for chemotaxis

2010 ◽  
Vol 190 (2) ◽  
pp. 233-245 ◽  
Author(s):  
Huaqing Cai ◽  
Satarupa Das ◽  
Yoichiro Kamimura ◽  
Yu Long ◽  
Carole A. Parent ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Time Course ◽  
Specific Inhibitor ◽  
Ras Proteins ◽  
Extracellular Signaling ◽  
Upstream Regulator ◽  
Pkb Phosphorylation ◽  
G Protein Coupled

In chemotactic cells, G protein–coupled receptors activate Ras proteins, but it is unclear how Ras-associated pathways link extracellular signaling to cell migration. We show that, in Dictyostelium discoideum, activated forms of RasC prolong the time course of TORC2 (target of rapamycin [Tor] complex 2)-mediated activation of a myristoylated protein kinase B (PKB; PKBR1) and the phosphorylation of PKB substrates, independently of phosphatidylinositol-(3,4,5)-trisphosphate. Paralleling these changes, the kinetics of chemoattractant-induced adenylyl cyclase activation and actin polymerization are extended, pseudopodial activity is increased and mislocalized, and chemotaxis is impaired. The effects of activated RasC are suppressed by deletion of the TORC2 subunit PiaA. In vitro RasCQ62L-dependent PKB phosphorylation can be rapidly initiated by the addition of a PiaA-associated immunocomplex to membranes of TORC2-deficient cells and blocked by TOR-specific inhibitor PP242. Furthermore, TORC2 binds specifically to the activated form of RasC. These results demonstrate that RasC is an upstream regulator of TORC2 and that the TORC2–PKB signaling mediates effects of activated Ras proteins on the cytoskeleton and cell migration.

scholarly journals Contribution of Filopodia to Cell Migration: A Mechanical Link between Protrusion and Contraction

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Fei Xue ◽  
Deanna M. Janzen ◽  
David A. Knecht
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Temporal Distribution ◽  
Leading Edge ◽  
Distal Portion ◽  
Actin Binding ◽  
Actin Networks ◽  
Motile Cells ◽  
A Cell

Numerous F-actin containing structures are involved in regulating protrusion of membrane at the leading edge of motile cells. We have investigated the structure and dynamics of filopodia as they relate to events at the leading edge and the function of the trailing actin networks. We have found that although filopodia contain parallel bundles of actin, they contain a surprisingly nonuniform spatial and temporal distribution of actin binding proteins. Along the length of the actin filaments in a single filopodium, the most distal portion contains primarily T-plastin, while the proximal portion is primarily bound byα-actinin and coronin. Some filopodia are stationary, but lateral filopodia move with respect to the leading edge. They appear to form a mechanical link between the actin polymerization network at the front of the cell and the myosin motor activity in the cell body. The direction of lateral filopodial movement is associated with the direction of cell migration. When lateral filopodia initiate from and move toward only one side of a cell, the cell will turn opposite to the direction of filopodial flow. Therefore, this filopodia-myosin II system allows actin polymerization driven protrusion forces and myosin II mediated contractile force to be mechanically coordinated.

scholarly journals Elasticity of dense actin networks produces nanonewton protrusive forces

2021 ◽  
Author(s):  
Marion Jasnin ◽  
Jordan Hervy ◽  
Stéphanie Balor ◽  
Anais Bouissou ◽  
Amsha Proag ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Actin Polymerization ◽  
Electron Tomography ◽  
Force Production ◽  
Basal Membrane ◽  
Theoretical Modelling ◽  
Elastic Behavior ◽  
Integrative Approach ◽  
Cellular Functions ◽  
Actin Networks

AbstractActin filaments assemble into force-generating systems involved in diverse cellular functions, including cell motility, adhesion, contractility and division. It remains unclear how networks of actin filaments, which individually generate piconewton forces, can produce forces reaching tens of nanonewtons. Here we use in situ cryo-electron tomography to unveil how the nanoscale architecture of macrophage podosomes enables basal membrane protrusion. We show that the sum of the actin polymerization forces at the membrane is not sufficient to explain podosome protrusive forces. Quantitative analysis of podosome organization demonstrates that the core is composed of a dense network of bent actin filaments storing elastic energy. Theoretical modelling of the network as a spring-loaded elastic material reveals that it exerts forces of up to tens of nanonewtons, similar to those evaluated experimentally. Thus, taking into account not only the interface with the membrane but also the bulk of the network, is crucial to understand force generation by actin machineries. Our integrative approach sheds light on the elastic behavior of dense actin networks and opens new avenues to understand force production inside cells.

F-actin serves as a template for cytokeratin organization in cell free extracts

2002 ◽  
Vol 115 (7) ◽  
pp. 1373-1382 ◽  
Author(s):  
Kari L. Weber ◽  
William M. Bement
Keyword(s):  
Time Course ◽  
Early Time ◽  
Dynamic Imaging ◽  
Actin Network ◽  
Intact Cells ◽  
Time Points ◽  
Egg Extracts ◽  
Actin Cables

The microtubule, F-actin, and intermediate filament systems are often studied as isolated systems, yet the three display mutual interdependence in living cells. To overcome limitations inherent in analysis of polymer-polymer interactions in intact cells, associations between these systems were assessed in Xenopus egg extracts. In both fixed and unfixed extract preparations, cytokeratin associated with F-actin cables that spontaneously assembled in the extracts. Time-course experiments revealed that at early time points cytokeratin cables were invariably associated with F-actin cables,while at later time points they could be found without associated F-actin. In extract samples where F-actin assembly was prevented, cytokeratin formed unorganized aggregates rather than cables. Dynamic imaging revealed transport of cytokeratin by moving F-actin as well as examples of cytokeratin release from F-actin. Experimental alteration of F-actin network organization by addition of α-actinin resulted in a corresponding change in the organization of the cytokeratin network. Finally, pharmacological disruption of the F-actin network in intact, activated eggs disrupted the normal pattern of cytokeratin assembly. These results provide direct evidence for an association between F-actin and cytokeratin in vitro and in vivo, and indicate that this interaction is necessary for proper cytokeratin assembly after transition into the first mitotic interphase of Xenopus.

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