scholarly journals Coupling of dynamic microtubules to F-actin by Fmn2 regulates chemotaxis of neuronal growth cones

2021 ◽  
Author(s):  
Tanushree Kundu ◽  
Priyanka Dutta ◽  
Dhriti Nagar ◽  
Sankar Maiti ◽  
Aurnab Ghose
Keyword(s):  
Single Molecule ◽  
Actin Filaments ◽  
Growth Cones ◽  
Axonal Pathfinding ◽  
Spinal Neurons ◽  
Critical Function ◽  
Superresolution Imaging ◽  
Cellular Processes ◽  
Neuronal Growth Cones

Dynamic co-regulation of the actin and microtubule subsystems enables the highly precise and adaptive remodelling of the cytoskeleton necessary for critical cellular processes, like axonal pathfinding. The modes and mediators of this interpolymer crosstalk, however, are inadequately understood. We identify Fmn2, a non-diaphanous related formin associated with cognitive disabilities, as a novel regulator of cooperative actin-microtubule remodelling in growth cones. We show that Fmn2 stabilizes microtubules in the growth cones of cultured spinal neurons and also in vivo. Superresolution imaging revealed that Fmn2 facilitates guidance of exploratory microtubules along actin bundles into the chemosensory filopodia. Using live imaging, biochemistry and single-molecule assays we show that a C-terminal domain in Fmn2 is necessary for the dynamic association between microtubules and actin filaments. In the absence of the cross- bridging function of Fmn2, filopodial capture of microtubules is compromised resulting in de-stabilized filopodial protrusions and deficits in growth cone chemotaxis. Our results uncover a critical function for Fmn2 in actin-microtubule crosstalk in neurons and demonstrate that modulating microtubule dynamics via associations with F-actin is central to directional motility.

scholarly journals Coupling of dynamic microtubules to F-actin by Fmn2 regulates chemotaxis of neuronal growth cones

2020 ◽  
Author(s):  
Tanushree Kundu ◽  
Priyanka Dutta ◽  
Dhriti Nagar ◽  
Sankar Maiti ◽  
Aurnab Ghose
Keyword(s):  
Single Molecule ◽  
Growth Cone ◽  
Growth Cones ◽  
Spinal Neurons ◽  
Critical Function ◽  
Superresolution Imaging ◽  
Cellular Processes ◽  
Component Systems ◽  
Cellular Behaviour

ABSTRACTDynamic co-regulation of the actin and microtubule subsystems enables the highly precise and adaptive remodeling of the cytoskeleton necessary for critical cellular processes, like axonal pathfinding. The modes and mediators of this interpolymer crosstalk, however, are inadequately understood.We identify Fmn2, a non-diaphanous related formin associated with cognitive disabilities, as a novel regulator of cooperative actin-microtubule remodeling in growth cones. We show that Fmn2 stabilizes microtubules in the growth cones of cultured spinal neurons and also in vivo. Superresolution imaging revealed that Fmn2 facilitates guidance of exploratory microtubules along actin bundles into the chemosensory filopodia. Using live imaging, biochemistry and single-molecule assays we show that a C-terminal domain in Fmn2 is necessary for the dynamic association between microtubules and actin filaments. In the absence of the cross-bridging function of Fmn2, filopodial capture of microtubules is compromised resulting in de-stabilized filopodial protrusions and deficits in growth cone chemotaxis.Our results uncover a critical function for Fmn2 in actin-microtubule crosstalk in neurons and demonstrate that modulating microtubule dynamics via associations with F-actin is central to directional motility.SIGNIFICANCE STATEMENTThe formin family member, Fmn2, is associated with cognitive impairment and neurodegenerative conditions though its function in neurons is poorly characterized. We report a novel actin-microtubule cross-bridging activity for Fmn2 that facilitates efficient targeting and capture of microtubules in growth cone filopodia. This activity is necessary for accurate pathfinding of axons and may contribute to Fmn2-associated neuropathologies.The precision and adaptability of cytoskeleton-driven processes are intimately dependent on the coupled activities of its component systems. Our study identifies a novel modality of co-regulated remodelling of the actin and microtubule cytoskeletons that facilitate critical cellular behaviour like neuronal chemotaxis.

scholarly journals Vesicle Size Regulates Nanotube Formation in the Cell

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Qian Peter Su ◽  
Wanqing Du ◽  
Qinghua Ji ◽  
Boxin Xue ◽  
Dong Jiang ◽  
...  
Keyword(s):  
Single Molecule ◽  
Model Systems ◽  
Organelle Biogenesis ◽  
Membrane Deformation ◽  
Vesicle Size ◽  
Cellular Processes ◽  
Vesicle Recycling ◽  
Physiological Relevance

Abstract Intracellular membrane nanotube formation and its dynamics play important roles for cargo transportation and organelle biogenesis. Regarding the regulation mechanisms, while much attention has been paid on the lipid composition and its associated protein molecules, effects of the vesicle size has not been studied in the cell. Giant unilamellar vesicles (GUVs) are often used for in vitro membrane deformation studies, but they are much larger than most intracellular vesicles and the in vitro studies also lack physiological relevance. Here, we use lysosomes and autolysosomes, whose sizes range between 100 nm and 1 μm, as model systems to study the size effects on nanotube formation both in vivo and in vitro. Single molecule observations indicate that driven by kinesin motors, small vesicles (100–200 nm) are mainly transported along the tracks while a remarkable portion of large vesicles (500–1000 nm) form nanotubes. This size effect is further confirmed by in vitro reconstitution assays on liposomes and purified lysosomes and autolysosomes. We also apply Atomic Force Microscopy (AFM) to measure the initiation force for nanotube formation. These results suggest that the size-dependence may be one of the mechanisms for cells to regulate cellular processes involving membrane-deformation, such as the timing of tubulation-mediated vesicle recycling.

scholarly journals A Slipping Clutch in Neuronal Growth Cones Revealed by Transient Single Molecule Interactions between Flowing Actin and N-Cadherin Adhesions

2014 ◽  
Vol 106 (2) ◽  
pp. 357a
Author(s):  
Mikael Garcia ◽  
Cécile Leduc ◽  
Amélie Argento ◽  
Jean-Baptiste Sibarita ◽  
Olivier R. Thoumine
Keyword(s):  
Single Molecule ◽  
Growth Cones ◽  
Neuronal Growth ◽  
Molecule Interactions ◽  
Neuronal Growth Cones

scholarly journals Watching cellular machinery in action, one molecule at a time

2016 ◽  
Vol 216 (1) ◽  
pp. 41-51 ◽  
Author(s):  
Enrico Monachino ◽  
Lisanne M. Spenkelink ◽  
Antoine M. van Oijen
Keyword(s):  
Dynamic Behavior ◽  
Single Molecule ◽  
Protein Complexes ◽  
Imaging Techniques ◽  
Ensemble Averaging ◽  
Cellular Processes ◽  
Cellular Machinery

Single-molecule manipulation and imaging techniques have become important elements of the biologist’s toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components.

scholarly journals Microtubule-associated Protein 2c Reorganizes Both Microtubules and Microfilaments into Distinct Cytological Structures in an Actin-binding Protein-280–deficient Melanoma Cell Line

1997 ◽  
Vol 136 (4) ◽  
pp. 845-857 ◽  
Author(s):  
C. Casey Cunningham ◽  
Nicole Leclerc ◽  
Lisa A. Flanagan ◽  
Mei Lu ◽  
Paul A. Janmey ◽  
...  
Keyword(s):  
Melanoma Cell ◽  
Binding Protein ◽  
Growth Cones ◽  
Actin Binding ◽  
Actin Binding Protein ◽  
Low Concentrations ◽  
Rheologic Property ◽  
Neuronal Growth Cones

The emergence of processes from cells often involves interactions between microtubules and microfilaments. Interactions between these two cytoskeletal systems are particularly apparent in neuronal growth cones. The juvenile isoform of the neuronal microtubule-associated protein 2 (MAP2c) is present in growth cones, where we hypothesize it mediates interactions between microfilaments and microtubules. To approach this problem in vivo, we used the human melanoma cell, M2, which lacks actin-binding protein-280 (ABP-280) and forms membrane blebs, which are not seen in wild-type or ABP-transfected cells. The microinjection of tau or mature MAP2 rescued the blebbing phenotype; MAP2c not only caused cessation of blebbing but also induced the formation of two distinct cellular structures. These were actin-rich lamellae, which often included membrane ruffles, and microtubule-bearing processes. The lamellae collapsed after treatment with cytochalasin D, and the processes retracted after treatment with colchicine. MAP2c was immunocytochemically visualized in zones of the cell that were devoid of tubulin, such as regions within the lamellae and in association with membrane ruffles. In vitro rheometry confirmed that MAP2c is an efficient actin gelation protein capable of organizing actin filaments into an isotropic array at very low concentrations; tau and mature MAP2 do not share this rheologic property. These results suggest that MAP2c engages in functionally specific interactions not only with microtubules but also with microfilaments.

scholarly journals Actin kinetics shapes cortical network structure and mechanics

2016 ◽  
Vol 2 (4) ◽  
pp. e1501337 ◽  
Author(s):  
Marco Fritzsche ◽  
Christoph Erlenkämper ◽  
Emad Moeendarbary ◽  
Guillaume Charras ◽  
Karsten Kruse
Keyword(s):  
Single Molecule ◽  
Actin Filaments ◽  
Length Distribution ◽  
Living Cells ◽  
Stochastic Simulations ◽  
Cortical Actin ◽  
Animal Cells ◽  
Cellular Mechanics ◽  
Cellular Processes ◽  
Actin Cortex

The actin cortex of animal cells is the main determinant of cellular mechanics. The continuous turnover of cortical actin filaments enables cells to quickly respond to stimuli. Recent work has shown that most of the cortical actin is generated by only two actin nucleators, the Arp2/3 complex and the formin Diaph1. However, our understanding of their interplay, their kinetics, and the length distribution of the filaments that they nucleate within living cells is poor. Such knowledge is necessary for a thorough comprehension of cellular processes and cell mechanics from basic polymer physics principles. We determined cortical assembly rates in living cells by using single-molecule fluorescence imaging in combination with stochastic simulations. We find that formin-nucleated filaments are, on average, 10 times longer than Arp2/3-nucleated filaments. Although formin-generated filaments represent less than 10% of all actin filaments, mechanical measurements indicate that they are important determinants of cortical elasticity. Tuning the activity of actin nucleators to alter filament length distribution may thus be a mechanism allowing cells to adjust their macroscopic mechanical properties to their physiological needs.

Transmembrane grasshopper Semaphorin I promotes axon outgrowth in vivo

Development ◽  
1997 ◽  
Vol 124 (18) ◽  
pp. 3597-3607 ◽  
Author(s):  
J.T. Wong ◽  
W.T. Yu ◽  
T.P. O'Connor
Keyword(s):  
Growth Cones ◽  
Limb Bud ◽  
Axonal Outgrowth ◽  
Axonal Pathfinding ◽  
Organ Growth ◽  
Guidance Cues ◽  
Alternative Function ◽  
Proximal Extension ◽  
Subgenual Organ

Members of the Semaphorin family of glycoproteins play an important role in axonal pathfinding by functioning as inhibitory guidance cues. Here we provide evidence that a transmembrane form of Semaphorin (Semaphorin I), which is expressed by bands of epithelial cells in the developing grasshopper limb bud, functions as an attractive/permissive cue for the growth cones of the subgenual organ. In addition, we demonstrate that Semaphorin I is needed for initial axonal outgrowth from the subgenual organ. These results are consistent with an alternative function for a transmembrane form of Semaphorin and may explain the previously reported arrest of the proximal extension of the subgenual organ growth cones in the absence of the Ti1 pioneer pathway.

scholarly journals Enhancement of Actin-depolymerizing Factor/Cofilin-dependent Actin Disassembly by Actin-interacting Protein 1 Is Required for Organized Actin Filament Assembly in the Caenorhabditis elegans Body Wall Muscle

2006 ◽  
Vol 17 (5) ◽  
pp. 2190-2199 ◽  
Author(s):  
Kurato Mohri ◽  
Kanako Ono ◽  
Robinson Yu ◽  
Sawako Yamashiro ◽  
Shoichiro Ono
Keyword(s):  
Actin Filament ◽  
Actin Filaments ◽  
Muscle Cells ◽  
Actin Depolymerizing Factor ◽  
Interacting Protein ◽  
Cellular Processes ◽  
Cytoskeletal Dynamics ◽  
End Capping

Regulated disassembly of actin filaments is involved in several cellular processes that require dynamic rearrangement of the actin cytoskeleton. Actin-interacting protein (AIP) 1 specifically enhances disassembly of actin-depolymerizing factor (ADF)/cofilin-bound actin filaments. In vitro, AIP1 actively disassembles filaments, caps barbed ends, and binds to the side of filaments. However, how AIP1 functions in the cellular actin cytoskeletal dynamics is not understood. We compared biochemical and in vivo activities of mutant UNC-78 proteins and found that impaired activity of mutant UNC-78 proteins to enhance disassembly of ADF/cofilin-bound actin filaments is associated with inability to regulate striated organization of actin filaments in muscle cells. Six functionally important residues are present in the N-terminal β-propeller, whereas one residue is located in the C-terminal β-propeller, suggesting the presence of two separate sites for interaction with ADF/cofilin and actin. In vitro, these mutant UNC-78 proteins exhibited variable alterations in actin disassembly and/or barbed end-capping activities, suggesting that both activities are important for its in vivo function. These results indicate that the actin-regulating activity of AIP1 in cooperation with ADF/cofilin is essential for its in vivo function to regulate actin filament organization in muscle cells.

scholarly journals The actin-binding ERM protein Moesin binds to and stabilizes microtubules at the cell cortex

2013 ◽  
Vol 202 (2) ◽  
pp. 251-260 ◽  
Author(s):  
Sara Solinet ◽  
Kazi Mahmud ◽  
Shannon F. Stewman ◽  
Khaled Ben El Kadhi ◽  
Barbara Decelle ◽  
...  
Keyword(s):  
Actin Filaments ◽  
Metastatic Potential ◽  
Actin Binding ◽  
Cell Cortex ◽  
Shape Transformation ◽  
Erm Proteins ◽  
Cellular Processes ◽  
Anaphase Onset

Ezrin, Radixin, and Moesin (ERM) proteins play important roles in many cellular processes including cell division. Recent studies have highlighted the implications of their metastatic potential in cancers. ERM’s role in these processes is largely attributed to their ability to link actin filaments to the plasma membrane. In this paper, we show that the ERM protein Moesin directly binds to microtubules in vitro and stabilizes microtubules at the cell cortex in vivo. We identified two evolutionarily conserved residues in the FERM (4.1 protein and ERM) domains of ERMs that mediated the association with microtubules. This ERM–microtubule interaction was required for regulating spindle organization in metaphase and cell shape transformation after anaphase onset but was dispensable for bridging actin filaments to the metaphase cortex. These findings provide a molecular framework for understanding the complex functional interplay between the microtubule and actin cytoskeletons mediated by ERM proteins in mitosis and have broad implications in both physiological and pathological processes that require ERMs.

Sign in / Sign up

Export Citation Format

Share Document