scholarly journals Src and cortactin promote lamellipodia protrusion and filopodia formation and stability in growth cones

2015 ◽  
Vol 26 (18) ◽  
pp. 3229-3244 ◽  
Author(s):  
Yingpei He ◽  
Yuan Ren ◽  
Bingbing Wu ◽  
Boris Decourt ◽  
Aih Cheun Lee ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Growth Cones ◽  
Network Density ◽  
Retrograde Flow ◽  
Actin Assembly ◽  
Actin Network ◽  
Cellular Mechanisms ◽  
Down Regulation ◽  
Actin Organization ◽  
Neuronal Growth Cones

Src tyrosine kinases have been implicated in axonal growth and guidance; however, the underlying cellular mechanisms are not well understood. Specifically, it is unclear which aspects of actin organization and dynamics are regulated by Src in neuronal growth cones. Here, we investigated the function of Src2 and one of its substrates, cortactin, in lamellipodia and filopodia of Aplysia growth cones. We found that up-regulation of Src2 activation state or cortactin increased lamellipodial length, protrusion time, and actin network density, whereas down-regulation had opposite effects. Furthermore, Src2 or cortactin up-regulation increased filopodial density, length, and protrusion time, whereas down-regulation promoted lateral movements of filopodia. Fluorescent speckle microscopy revealed that rates of actin assembly and retrograde flow were not affected in either case. In summary, our results support a model in which Src and cortactin regulate growth cone motility by increasing actin network density and protrusion persistence of lamellipodia by controlling the state of actin-driven protrusion versus retraction. In addition, both proteins promote the formation and stability of actin bundles in filopodia.

scholarly journals Protein kinase C activation decreases peripheral actin network density and increases central nonmuscle myosin II contractility in neuronal growth cones

2013 ◽  
Vol 24 (19) ◽  
pp. 3097-3114 ◽  
Author(s):  
Qing Yang ◽  
Xiao-Feng Zhang ◽  
David Van Goor ◽  
Ashleigh P. Dunn ◽  
Callen Hyland ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Myosin Ii ◽  
Growth Cones ◽  
Network Density ◽  
Actin Network ◽  
Neuronal Growth ◽  
Kinase C ◽  
Neuronal Growth Cones ◽  
Pkc Activation

Protein kinase C (PKC) can dramatically alter cell structure and motility via effects on actin filament networks. In neurons, PKC activation has been implicated in repulsive guidance responses and inhibition of axon regeneration; however, the cytoskeletal mechanisms underlying these effects are not well understood. Here we investigate the acute effects of PKC activation on actin network structure and dynamics in large Aplysia neuronal growth cones. We provide evidence of a novel two-tiered mechanism of PKC action: 1) PKC activity enhances myosin II regulatory light chain phosphorylation and C-kinase–potentiated protein phosphatase inhibitor phosphorylation. These effects are correlated with increased contractility in the central cytoplasmic domain. 2) PKC activation results in significant reduction of P-domain actin network density accompanied by Arp2/3 complex delocalization from the leading edge and increased rates of retrograde actin network flow. Our results show that PKC activation strongly affects both actin polymerization and myosin II contractility. This synergistic mode of action is relevant to understanding the pleiotropic reported effects of PKC on neuronal growth and regeneration.

scholarly journals L1-dependent neuritogenesis involves ankyrinB that mediates L1-CAM coupling with retrograde actin flow

2003 ◽  
Vol 163 (5) ◽  
pp. 1077-1088 ◽  
Author(s):  
Kazunari Nishimura ◽  
Fumie Yoshihara ◽  
Takuro Tojima ◽  
Noriko Ooashi ◽  
Woohyun Yoon ◽  
...  
Keyword(s):  
Cell Adhesion Molecule ◽  
Traction Force ◽  
Neurite Growth ◽  
Growth Cones ◽  
Retrograde Flow ◽  
Actin Network ◽  
Filamentous Actin ◽  
Neurite Elongation ◽  
Neurite Initiation ◽  
Cell Adhesion Molecule L1

The cell adhesion molecule L1 (L1-CAM) plays critical roles in neurite growth. Its cytoplasmic domain (L1CD) binds to ankyrins that associate with the spectrin–actin network. This paper demonstrates that L1-CAM interactions with ankyrinB (but not with ankyrinG) are involved in the initial formation of neurites. In the membranous protrusions surrounding the soma before neuritogenesis, filamentous actin (F-actin) and ankyrinB continuously move toward the soma (retrograde flow). Bead-tracking experiments show that ankyrinB mediates L1-CAM coupling with retrograde F-actin flow in these perisomatic structures. Ligation of the L1-CAM ectodomain by an immobile substrate induces L1CD–ankyrinB binding and the formation of stationary ankyrinB clusters. Neurite initiation preferentially occurs at the site of these clusters. In contrast, ankyrinB is involved neither in L1-CAM coupling with F-actin flow in growth cones nor in L1-based neurite elongation. Our results indicate that ankyrinB promotes neurite initiation by acting as a component of the clutch module that transmits traction force generated by F-actin flow to the extracellular substrate via L1-CAM.

scholarly journals Tracking Retrograde Flow in Keratocytes: News from the Front

2005 ◽  
Vol 16 (3) ◽  
pp. 1223-1231 ◽  
Author(s):  
Pascal Vallotton ◽  
Gaudenz Danuser ◽  
Sophie Bohnet ◽  
Jean-Jacques Meister ◽  
Alexander B. Verkhovsky
Keyword(s):  
Actin Polymerization ◽  
Leading Edge ◽  
Membrane Resistance ◽  
Retrograde Flow ◽  
Actin Assembly ◽  
Actin Network ◽  
Freely Moving ◽  
Contractile Forces ◽  
Cell Motion ◽  
Shape Changes

Actin assembly at the leading edge of the cell is believed to drive protrusion, whereas membrane resistance and contractile forces result in retrograde flow of the assembled actin network away from the edge. Thus, cell motion and shape changes are expected to depend on the balance of actin assembly and retrograde flow. This idea, however, has been undermined by the reported absence of flow in one of the most spectacular models of cell locomotion, fish epidermal keratocytes. Here, we use enhanced phase contrast and fluorescent speckle microscopy and particle tracking to analyze the motion of the actin network in keratocyte lamellipodia. We have detected retrograde flow throughout the lamellipodium at velocities of 1–3 μm/min and analyzed its organization and relation to the cell motion during both unobstructed, persistent migration and events of cell collision. Freely moving cells exhibited a graded flow velocity increasing toward the sides of the lamellipodium. In colliding cells, the velocity decreased markedly at the site of collision, with striking alteration of flow in other lamellipodium regions. Our findings support the universality of the flow phenomenon and indicate that the maintenance of keratocyte shape during locomotion depends on the regulation of both retrograde flow and actin polymerization.

scholarly journals Xenopus cytoplasmic linker–associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules

2013 ◽  
Vol 24 (10) ◽  
pp. 1544-1558 ◽  
Author(s):  
Astrid Marx ◽  
William J. Godinez ◽  
Vasil Tsimashchuk ◽  
Peter Bankhead ◽  
Karl Rohr ◽  
...  
Keyword(s):  
Growth Cone ◽  
Binding Protein ◽  
Leading Edge ◽  
Growth Cones ◽  
Retrograde Flow ◽  
Spinal Cord Neurons ◽  
Actin Organization ◽  
Axon Elongation ◽  
Axon Extension ◽  
Potential Link

Dynamic microtubules (MTs) are required for neuronal guidance, in which axons extend directionally toward their target tissues. We found that depletion of the MT-binding protein Xenopus cytoplasmic linker–associated protein 1 (XCLASP1) or treatment with the MT drug Taxol reduced axon outgrowth in spinal cord neurons. To quantify the dynamic distribution of MTs in axons, we developed an automated algorithm to detect and track MT plus ends that have been fluorescently labeled by end-binding protein 3 (EB3). XCLASP1 depletion reduced MT advance rates in neuronal growth cones, very much like treatment with Taxol, demonstrating a potential link between MT dynamics in the growth cone and axon extension. Automatic tracking of EB3 comets in different compartments revealed that MTs increasingly slowed as they passed from the axon shaft into the growth cone and filopodia. We used speckle microscopy to demonstrate that MTs experience retrograde flow at the leading edge. Microtubule advance in growth cone and filopodia was strongly reduced in XCLASP1-depleted axons as compared with control axons, but actin retrograde flow remained unchanged. Instead, we found that XCLASP1-depleted growth cones lacked lamellipodial actin organization characteristic of protrusion. Lamellipodial architecture depended on XCLASP1 and its capacity to associate with MTs, highlighting the importance of XCLASP1 in actin–microtubule interactions.

scholarly journals Wnt5A-Mediated Actin Organization Regulates Host Response to Bacterial Pathogens and Non-Pathogens

2021 ◽  
Vol 11 ◽  
Author(s):  
Suborno Jati ◽  
Soham Sengupta ◽  
Malini Sen
Keyword(s):  
Conditioned Medium ◽  
Host Response ◽  
Bacterial Pathogens ◽  
The State ◽  
Actin Assembly ◽  
Actin Network ◽  
Actin Organization ◽  
E Coli ◽  
Interspecies Variability

Wnt5A signaling facilitates the killing of several bacterial pathogens, but not the non-pathogen E. coli DH5α. The basis of such pathogen vs. non-pathogen distinction is unclear. Accordingly, we analyzed the influence of Wnt5A signaling on pathogenic E. coli K1 in relation to non-pathogenic E. coli K12-MG1655 and E. coli DH5α eliminating interspecies variability from our study. Whereas cell internalized E. coli K1 disrupted cytoskeletal actin organization and multiplied during Wnt5A depletion, rWnt5A mediated activation revived cytoskeletal actin assembly facilitating K1 eradication. Cell internalized E. coli K12-MG1655 and E. coli DH5α, which did not perturb actin assembly appreciably, remained unaffected by rWnt5A treatment. Phagosomes prepared separately from Wnt5A conditioned medium treated K1 and K12-MG1655 infected macrophages revealed differences in the relative levels of actin and actin network promoting proteins, upholding that the Wnt5A-Actin axis operates differently for internalized pathogen and non-pathogen. Interestingly, exposure of rWnt5A treated K1 and K12-MG1655/DH5α infected macrophages to actin assembly inhibitors reversed the scenario, blocking killing of K1, yet promoting killing of both K12-MG1655 and DH5α. Taken together, our study illustrates that the state of activation of the Wnt5A/Actin axis in the context of the incumbent bacteria is crucial for directing host response to infection.

scholarly journals A dynamic formin-dependent deep F-actin network in axons

2015 ◽  
Vol 210 (3) ◽  
pp. 401-417 ◽  
Author(s):  
Archan Ganguly ◽  
Yong Tang ◽  
Lina Wang ◽  
Kelsey Ladt ◽  
Jonathan Loi ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Super Resolution ◽  
Growth Cones ◽  
Filamentous Actin ◽  
Actin Organization ◽  
Cytoskeletal Network ◽  
Presynaptic Boutons ◽  
Super Resolution Microscopy ◽  
Neuronal Growth Cones ◽  
Actin Rings

Although actin at neuronal growth cones is well-studied, much less is known about actin organization and dynamics along axon shafts and presynaptic boutons. Using probes that selectively label filamentous-actin (F-actin), we found focal “actin hotspots” along axons—spaced ∼3–4 µm apart—where actin undergoes continuous assembly/disassembly. These foci are a nidus for vigorous actin polymerization, generating long filaments spurting bidirectionally along axons—a phenomenon we call “actin trails.” Super-resolution microscopy reveals intra-axonal deep actin filaments in addition to the subplasmalemmal “actin rings” described recently. F-actin hotspots colocalize with stationary axonal endosomes, and blocking vesicle transport diminishes the actin trails, suggesting mechanistic links between vesicles and F-actin kinetics. Actin trails are formin—but not Arp2/3—dependent and help enrich actin at presynaptic boutons. Finally, formin inhibition dramatically disrupts synaptic recycling. Collectively, available data suggest a two-tier F-actin organization in axons, with stable “actin rings” providing mechanical support to the plasma membrane and dynamic "actin trails" generating a flexible cytoskeletal network with putative physiological roles.

scholarly journals The Role of Actin Turnover in Retrograde Actin Network Flow in Neuronal Growth Cones

PLoS ONE ◽  
2012 ◽  
Vol 7 (2) ◽  
pp. e30959 ◽  
Author(s):  
David Van Goor ◽  
Callen Hyland ◽  
Andrew W. Schaefer ◽  
Paul Forscher
Keyword(s):  
Network Flow ◽  
Growth Cones ◽  
Actin Network ◽  
Neuronal Growth ◽  
Actin Turnover ◽  
Neuronal Growth Cones

scholarly journals Filopodia and actin arcs guide the assembly and transport of two populations of microtubules with unique dynamic parameters in neuronal growth cones

2002 ◽  
Vol 158 (1) ◽  
pp. 139-152 ◽  
Author(s):  
Andrew W. Schaefer ◽  
Nurul Kabir ◽  
Paul Forscher
Keyword(s):  
Dynamic Instability ◽  
Growth Cones ◽  
Polymer Dynamics ◽  
Differential Interference Contrast ◽  
Retrograde Flow ◽  
Contrast Imaging ◽  
P Domain ◽  
Neuronal Growth Cones ◽  
Two Populations ◽  
Peripheral Domain

We have used multimode fluorescent speckle microscopy (FSM) and correlative differential interference contrast imaging to investigate the actin–microtubule (MT) interactions and polymer dynamics known to play a fundamental role in growth cone guidance. We report that MTs explore the peripheral domain (P-domain), exhibiting classical properties of dynamic instability. MT extension occurs preferentially along filopodia, which function as MT polymerization guides. Filopodial bundles undergo retrograde flow and also transport MTs. Thus, distal MT position is determined by the rate of plus-end MT assembly minus the rate of retrograde F-actin flow. Short MT displacements independent of flow are sometimes observed. MTs loop, buckle, and break as they are transported into the T-zone by retrograde flow. MT breakage results in exposure of new plus ends which can regrow, and minus ends which rapidly undergo catastrophes, resulting in efficient MT turnover. We also report a previously undetected presence of F-actin arc structures, which exhibit persistent retrograde movement across the T-zone into the central domain (C-domain) at ∼1/4 the rate of P-domain flow. Actin arcs interact with MTs and transport them into the C-domain. Interestingly, although the MTs associated with arcs are less dynamic than P-domain MTs, they elongate efficiently as a result of markedly lower catastrophe frequencies.

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