scholarly journals Assembly and Activity of the WASH Molecular Machine: Distinctive Features at the Crossroads of the Actin and Microtubule Cytoskeletons

2021 ◽  
Vol 9 ◽  
Author(s):  
Artem I. Fokin ◽  
Alexis M. Gautreau
Keyword(s):  
Cell Cortex ◽  
Membrane Remodeling ◽  
Distinctive Features ◽  
Actin Networks ◽  
Capping Protein ◽  
Endosomal Membrane ◽  
Pulling Forces ◽  
Endosomal Membranes ◽  
Privileged Site ◽  
Pushing And Pulling

The Arp2/3 complex generates branched actin networks at different locations of the cell. The WASH and WAVE Nucleation Promoting Factors (NPFs) activate the Arp2/3 complex at the surface of endosomes or at the cell cortex, respectively. In this review, we will discuss how these two NPFs are controlled within distinct, yet related, multiprotein complexes. These complexes are not spontaneously assembled around WASH and WAVE, but require cellular assembly factors. The centrosome, which nucleates microtubules and branched actin, appears to be a privileged site for WASH complex assembly. The actin and microtubule cytoskeletons are both responsible for endosome shape and membrane remodeling. Motors, such as dynein, pull endosomes and extend membrane tubules along microtubule tracks, whereas branched actin pushes onto the endosomal membrane. It was recently uncovered that WASH assembles a super complex with dynactin, the major dynein activator, where the Capping Protein (CP) is exchanged from dynactin to the WASH complex. This CP swap initiates the first actin filament that primes the autocatalytic nucleation of branched actin at the surface of endosomes. Possible coordination between pushing and pulling forces in the remodeling of endosomal membranes is discussed.

scholarly journals ENDOSOMAL MEMBRANE TENSION CONTROLS ESCRT-III-DEPENDENT INTRA-LUMENAL VESICLE FORMATION

2019 ◽  
Author(s):  
Vincent Mercier ◽  
Jorge Larios ◽  
Guillaume Molinard ◽  
Antoine Goujon ◽  
Stefan Matile ◽  
...  
Keyword(s):  
Vesicle Formation ◽  
Membrane Tension ◽  
Membrane Remodeling ◽  
Intracellular Traffic ◽  
Physiological Conditions ◽  
Endosomal Membrane ◽  
Endosome Trafficking ◽  
Endosomal Membranes

AbstractPlasma membrane tension strongly affects cell surface processes, such as migration, endocytosis and signalling. However, it is not known whether membrane tension of organelles regulates their functions, notably intracellular traffic. The ESCRT-III complex is the major membrane remodelling complex that drives Intra-Lumenal Vesicle (ILV) formation on endosomal membranes. Here, we made use of a new fluorescent membrane tension probe to show that ESCRT-III subunits are recruited onto endosomal membranes when membrane tension is reduced. We find that tension-dependent recruitment is associated with ESCRT-III polymerization and membrane deformation in vitro, and correlates with increased ILVs formation in ESCRT-III decorated endosomes in vivo. Finally, we find that endosomal membrane tension decreases when ILV formation is triggered by EGF under physiological conditions. These results indicate that membrane tension is a major regulator of ILV formation and of endosome trafficking, leading us to conclude that membrane tension can control organelle functions.One Sentence SummaryMembrane tension decrease facilitates membrane remodeling by ESCRT-III polymerization during intra-lumenal vesicle formation.

scholarly journals Centrosome centering and decentering by microtubule network rearrangement

2016 ◽  
Vol 27 (18) ◽  
pp. 2833-2843 ◽  
Author(s):  
Gaëlle Letort ◽  
Francois Nedelec ◽  
Laurent Blanchoin ◽  
Manuel Théry
Keyword(s):  
Molecular Motors ◽  
Cell Polarization ◽  
Cell Cortex ◽  
Cell Periphery ◽  
Microtubule Network ◽  
Abrupt Transition ◽  
External Cues ◽  
Pulling Forces ◽  
Number Dynamics ◽  
Pushing And Pulling

The centrosome is positioned at the cell center by pushing and pulling forces transmitted by microtubules (MTs). Centrosome decentering is often considered to result from asymmetric, cortical pulling forces exerted in particular by molecular motors on MTs and controlled by external cues affecting the cell cortex locally. Here we used numerical simulations to investigate the possibility that it could equally result from the redistribution of pushing forces due to a reorientation of MTs. We first showed that MT gliding along cell edges and pivoting around the centrosome regulate MT rearrangement and thereby direct the spatial distribution of pushing forces, whereas the number, dynamics, and stiffness of MTs determine the magnitude of these forces. By modulating these parameters, we identified different regimes, involving both pushing and pulling forces, characterized by robust centrosome centering, robust off-centering, or “reactive” positioning. In the last-named conditions, weak asymmetric cues can induce a misbalance of pushing and pulling forces, resulting in an abrupt transition from a centered to an off-centered position. Taken together, these results point to the central role played by the configuration of the MTs on the distribution of pushing forces that position the centrosome. We suggest that asymmetric external cues should not be seen as direct driver of centrosome decentering and cell polarization but instead as inducers of an effective reorganization of the MT network, fostering centrosome motion to the cell periphery.

scholarly journals Centrosome centering and Decentering by microtubule network rearrangement

2016 ◽  
Author(s):  
Gaëlle Letort ◽  
Francois Nedelec ◽  
Laurent Blanchoin ◽  
Manuel Théry
Keyword(s):  
Molecular Motors ◽  
Cell Polarization ◽  
Cell Cortex ◽  
Cell Periphery ◽  
Microtubule Network ◽  
Abrupt Transition ◽  
External Cues ◽  
Pulling Forces ◽  
Number Dynamics ◽  
Pushing And Pulling

AbstractThe centrosome is positioned at the cell center by pushing and pulling forces transmitted by microtubules (MTs). Centrosome decentering is often considered to result from asymmetric, cortical pulling forces exerted in particular by molecular motors on MTs, and controlled by external cues affecting the cell cortex locally. Here we used numerical simulations to investigate the possibility that it could equally result from the redistribution of pushing forces due to a reorientation of MTs. We first showed that MT gliding along cell edges and pivoting around the centrosome regulate MT rearrangement and thereby direct the spatial distribution of pushing forces, while the number, dynamics and stiffness of MTs determine the magnitude of these forces. By modulating these parameters, we identified different regimes, involving both pushing and pulling forces, characterized either by robust centrosome centering, robust off-centering or “reactive” positioning. In those latter conditions weak asymmetric cues can induce a misbalance of pushing and pulling forces resulting in an abrupt transition from a centered to an off-centered position. Altogether these results point at the central role played by the configuration of the MTs on the distribution of pushing forces that position the centrosome. We suggest that asymmetric external cues should not be seen as direct driver of centrosome decentering and cell polarization, but rather as inducers of an effective reorganization of the MT network, fostering centrosome motion to the cell periphery.

scholarly journals Exosomes induce endolysosomal permeabilization as a gateway by which exosomal tau seeds escape into the cytosol

2021 ◽  
Author(s):  
Juan Carlos Polanco ◽  
Gabriel Rhys Hand ◽  
Adam Briner ◽  
Chuanzhou Li ◽  
Jürgen Götz
Keyword(s):  
Critical Role ◽  
Membrane Integrity ◽  
Lysosomal Degradation ◽  
Escape Route ◽  
Cellular Invasion ◽  
Endosomal Membrane ◽  
Late Endosomes ◽  
Endosomal Membranes ◽  
Endosomal Pathway ◽  
Tau Aggregation

AbstractThe microtubule-associated protein tau has a critical role in Alzheimer’s disease and other tauopathies. A proposed pathomechanism in the progression of tauopathies is the trans-synaptic spreading of tau seeds, with a role for exosomes which are secretory nanovesicles generated by late endosomes. Our previous work demonstrated that brain-derived exosomes isolated from tau transgenic rTg4510 mice encapsulate tau seeds with the ability to induce tau aggregation in recipient cells. We had also shown that exosomes can hijack the endosomal pathway to spread through interconnected neurons. Here, we reveal how tau seeds contained within internalized exosomes exploit mechanisms of lysosomal degradation to escape the endosome and induce tau aggregation in the cytosol of HEK293T-derived ‘tau biosensor cells’. We found that the majority of the exosome-containing endosomes fused with lysosomes to form endolysosomes. Exosomes induced their permeabilization, irrespective of the presence of tau seeds, or whether the exosomal preparations originated from mouse brains or HEK293T cells. We also found that permeabilization is a conserved mechanism, operating in both non-neuronal tau biosensor cells and primary neurons. However, permeabilization of endolysosomes only occurred in a small fraction of cells, which supports the notion that permeabilization occurs by a thresholded mechanism. Interestingly, tau aggregation was only induced in cells that exhibited permeabilization, presenting this as an escape route of exosomal tau seeds into the cytosol. Overexpression of RAB7, which is required for the formation of endolysosomes, strongly increased tau aggregation. Conversely, inhibition of lysosomal function with alkalinizing agents, or by knocking-down RAB7, decreased tau aggregation. Together, we conclude that the enzymatic activities of lysosomes permeabilize exosomal and endosomal membranes, thereby facilitating access of exosomal tau seeds to cytosolic tau to induce its aggregation. Our data underscore the importance of endosomal membrane integrity in mechanisms of cellular invasion by misfolded proteins that are resistant to lysosomal degradation.

scholarly journals The forces that position a mitotic spindle asymmetrically are tethered until after the time of spindle assembly

2004 ◽  
Vol 167 (2) ◽  
pp. 245-256 ◽  
Author(s):  
Jean-Claude Labbé ◽  
Erin K. McCarthy ◽  
Bob Goldstein
Keyword(s):  
Mitotic Spindle ◽  
Spindle Assembly ◽  
Cell Cortex ◽  
Par Proteins ◽  
Temporal Regulation ◽  
C Elegans ◽  
Pulling Forces ◽  
Chromosome Separation ◽  
And Shifts ◽  
Early Phases

Regulation of the mitotic spindle's position is important for cells to divide asymmetrically. Here, we use Caenorhabditis elegans embryos to provide the first analysis of the temporal regulation of forces that asymmetrically position a mitotic spindle. We find that asymmetric pulling forces, regulated by cortical PAR proteins, begin to act as early as prophase and prometaphase, even before the spindle forms and shifts to a posterior position. The spindle does not shift asymmetrically during these early phases due to a tethering force, mediated by astral microtubules that reach the anterior cell cortex. We show that this tether is normally released after spindle assembly and independently of anaphase entry. Monitoring microtubule dynamics by photobleaching segments of microtubules during anaphase revealed that spindle microtubules do not undergo significant poleward flux in C. elegans. Together with the known absence of anaphase A, these data suggest that the major forces contributing to chromosome separation during anaphase originate outside the spindle. We propose that the forces positioning the mitotic spindle asymmetrically are tethered until after the time of spindle assembly and that these same forces are used later to drive chromosome segregation at anaphase.

scholarly journals Positioning of microtubule organizing centers by cortical pushing and pulling forces

2012 ◽  
Vol 14 (10) ◽  
pp. 105025 ◽  
Author(s):  
Nenad Pavin ◽  
Liedewij Laan ◽  
Rui Ma ◽  
Marileen Dogterom ◽  
Frank Jülicher
Keyword(s):  
Microtubule Organizing Centers ◽  
Pulling Forces ◽  
Pushing And Pulling

scholarly journals Twinfilin uncaps filament barbed ends to promote turnover of lamellipodial actin networks

2019 ◽  
Author(s):  
Markku Hakala ◽  
Hugo Wioland ◽  
Mari Tolonen ◽  
Antoine Jegou ◽  
Guillaume Romet-Lemonne ◽  
...  
Keyword(s):  
Leading Edge ◽  
Underlying Mechanism ◽  
Actin Dynamics ◽  
Dissociation Kinetics ◽  
Single Filament ◽  
Actin Networks ◽  
Capping Protein ◽  
Specific Localization ◽  
In Cells

AbstractCoordinated polymerization of actin filaments provides force for cell migration, morphogenesis, and endocytosis. Capping Protein (CP) is central regulator of actin dynamics in all eukaryotes. It binds actin filament (F-actin) barbed ends with high affinity and slow dissociation kinetics to prevent filament polymerization and depolymerization. In cells, however, CP displays remarkably rapid dynamics within F-actin networks, but the underlying mechanism has remained enigmatic. We report that a conserved cytoskeletal regulator, twinfilin, is responsible for CP’s rapid dynamics and specific localization in cells. Depletion of twinfilin led to stable association of CP with cellular F-actin arrays and its treadmilling throughout leading-edge lamellipodium. These were accompanied by diminished F-actin disassembly rates. In vitro single filament imaging approaches revealed that twinfilin directly promotes dissociation of CP from filament barbed ends, while allowing subsequent filament depolymerization. These results uncover an evolutionary conserved bipartite mechanism that controls how actin cytoskeleton-mediated forces are generated in cells.

scholarly journals Overexpression of Mdm36 reveals Num1 foci that mediate dynein-dependent microtubule sliding in budding yeast

2020 ◽  
Author(s):  
Safia Omer ◽  
Katia Brock ◽  
John Beckford ◽  
Wei-Lih Lee
Keyword(s):  
Budding Yeast ◽  
Current Model ◽  
Cytoplasmic Dynein ◽  
Cell Cortex ◽  
Direct Imaging ◽  
Spindle Positioning ◽  
Sliding Mechanism ◽  
Pulling Forces ◽  
Pulling Force

ABSTRACTCurrent model for spindle positioning requires attachment of the microtubule (MT) motor cytoplasmic dynein to the cell cortex, where it generates pulling force on astral MTs to effect spindle displacement. How dynein is anchored by cortical attachment machinery to generate large spindle-pulling forces remains unclear. Here, we show that cortical clustering of Num1, the yeast dynein attachment molecule, is limited by Mdm36. Overexpression of Mdm36 results in an overall enhancement of Num1 clustering but reveals a population of dim Num1 clusters that mediate dynein-anchoring at the cell cortex. Direct imaging shows that bud-localized, dim Num1 clusters containing only ∼6 copies of Num1 molecules mediate dynein-dependent spindle pulling via lateral MT sliding mechanism. Mutations affecting Num1 clustering interfere with mitochondrial tethering but not dynein-based spindle-pulling function of Num1. We propose that formation of small ensembles of attachment molecules is sufficient for dynein anchorage and cortical generation of large spindle-pulling force.

scholarly journals The Arp1/11 minifilament of dynactin primes the endosomal Arp2/3 complex

2021 ◽  
Vol 7 (3) ◽  
pp. eabd5956 ◽  
Author(s):  
Artem I. Fokin ◽  
Violaine David ◽  
Ksenia Oguievetskaia ◽  
Emmanuel Derivery ◽  
Caroline E. Stone ◽  
...  
Keyword(s):  
Actin Filament ◽  
Actin Networks ◽  
Capping Protein ◽  
Related Proteins ◽  
In Cells

Dendritic actin networks develop from a first actin filament through branching by the Arp2/3 complex. At the surface of endosomes, the WASH complex activates the Arp2/3 complex and interacts with the capping protein for unclear reasons. Here, we show that the WASH complex interacts with dynactin and uncaps it through its FAM21 subunit. In vitro, the uncapped Arp1/11 minifilament elongates an actin filament, which then primes the WASH-induced Arp2/3 branching reaction. In dynactin-depleted cells or in cells where the WASH complex is reconstituted with a FAM21 mutant that cannot uncap dynactin, formation of branched actin at the endosomal surface is impaired. Our results reveal the importance of the WASH complex in coordinating two complexes containing actin-related proteins.

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