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The authors show how tubular surface structures in all cell types, have the ability to twist and perform rotary sweeping motion to explore the extracellular environment. This has implications for migration, sensing and cell communication.Filopodia are actin-rich structures, present on the surface of eukaryotic cells. These structures play a pivotal role by allowing cells to explore their environment, generate mechanical forces or perform chemical signaling. Their complex dynamics includes buckling, pulling, length and shape changes. We show that filopodia additionally explore their 3D extracellular space by combining growth and shrinking with axial twisting and buckling. Importantly, the actin core inside filopodia performs a twisting or spinning motion which is observed for a range of cell types spanning from earliest development to highly differentiated tissue cells. Non-equilibrium physical modeling of actin and myosin confirm that twist is an emergent phenomenon of active filaments confined in a narrow channel which is supported by measured traction forces and helical buckles that can be ascribed to accumulation of sufficient twist. These results lead us to conclude that activity induced twisting of the actin shaft is a general mechanism underlying fundamental functions of filopodia.

The authors show how tubular surface structures in all cell types, have the ability to twist and perform rotary sweeping motion to explore the extracellular environment. This has implications for migration, sensing and cell communication.Filopodia are actin-rich structures, present on the surface of eukaryotic cells. These structures play a pivotal role by allowing cells to explore their environment, generate mechanical forces or perform chemical signaling. Their complex dynamics includes buckling, pulling, length and shape changes. We show that filopodia additionally explore their 3D extracellular space by combining growth and shrinking with axial twisting and buckling. Importantly, the actin core inside filopodia performs a twisting or spinning motion which is observed for a range of cell types spanning from earliest development to highly differentiated tissue cells. Non-equilibrium physical modeling of actin and myosin confirm that twist is an emergent phenomenon of active filaments confined in a narrow channel which is supported by measured traction forces and helical buckles that can be ascribed to accumulation of sufficient twist. These results lead us to conclude that activity induced twisting of the actin shaft is a general mechanism underlying fundamental functions of filopodia.

Neurodegenerative diseases such as Alzheimer’s disease (AD) are becoming more prevalent as the average human lifespan increases. Despite decades of research, no therapeutics exist that can slow the progress of AD. One of the hallmark symptoms that best correlates with symptoms of AD is synapse loss at the cellular level. Ethylene glycol (EG) analogs of benzothiazole aniline (BTA) improve memory and learning in mice by increasing the number of postsynaptic connection sites or dendritic spines in neurons. These compounds interact with the protein Fascin1. Through a combination of knockdown and overexpression studies, I show that Fascin1 levels affect dendritic spine density. Using tandem mass tag mass spectrometry pulldowns using human brain cortex lysate, I identify Fascin1-protein interactions that change in the presence of BTA-EG4 and BTA-EG6. I identify changes in focal adhesions, or sites at which the cell creates an attachment to the extracellular matrix, by immunofluorescence as a method by which BTA-EG4 and BTA-EG6 may be impacting dendritic spine density. I also provide structural evidence for the binding pocket between Fascin1 and BTA-EG6 using solution protein nuclear magnetic resonance (NMR) and site-directed mutagenesis.

Neurodegenerative diseases such as Alzheimer’s disease (AD) are becoming more prevalent as the average human lifespan increases. Despite decades of research, no therapeutics exist that can slow the progress of AD. One of the hallmark symptoms that best correlates with symptoms of AD is synapse loss at the cellular level. Ethylene glycol (EG) analogs of benzothiazole aniline (BTA) improve memory and learning in mice by increasing the number of postsynaptic connection sites or dendritic spines in neurons. These compounds interact with the protein Fascin1. Through a combination of knockdown and overexpression studies, I show that Fascin1 levels affect dendritic spine density. Using tandem mass tag mass spectrometry pulldowns using human brain cortex lysate, I identify Fascin1-protein interactions that change in the presence of BTA-EG4 and BTA-EG6. I identify changes in focal adhesions, or sites at which the cell creates an attachment to the extracellular matrix, by immunofluorescence as a method by which BTA-EG4 and BTA-EG6 may be impacting dendritic spine density. I also provide structural evidence for the binding pocket between Fascin1 and BTA-EG6 using solution protein nuclear magnetic resonance (NMR) and site-directed mutagenesis.

Fascin-1 is an actin-bundling protein localized at the core actin bundles within microvillar projections and filopodial extensions in migrating cells. It is expressed at a low level in normal epithelial cells, but at a high level in tumor cells, indicating its importance in the invasion and progression of tumor cells. In addition, fascin-1 is expressed in human and murine embryos, especially at high levels in developing nervous tissues. In this study, we immunohistochemically investigated the expression patterns of fascin-1 during the early stages of rat hepatogenesis. A high expression of fascin-1 was detected in the liver bud and hepatoblasts at embryonic day (ED) 10.5, 11.5 and 12.5. The expression fell by ED13.5 and was not detectable at ED14.5. These observations demonstrated that the expression of fascin-1 is correlated with the migration activity of hepatoblasts during the early stages of liver development in rats., source:http://www.springerlink.com/content/r23363757t54u184

Background: Bundles of unipolar actin filaments (F-actin), cross-linked via the actin-binding protein fascin, are important in filopodia of motile cells and stereocilia of inner ear sensory cells. However, such bundles are also useful as shuttles in myosin-driven nanotechnological applications. Therefore, and for elucidating aspects of biological function, we investigate if the bundle tendency to follow straight paths (quantified by path persistence length) when propelled by myosin motors is directly determined by material properties quantified by persistence length of thermally fluctuating bundles. Methods: Fluorescent bundles, labeled with rhodamine-phalloidin, were studied at fascin:actin molar ratios: 0:1 (F-actin), 1:7, 1:4 and 1:2. Persistence lengths (Lp) were obtained by fitting the cosine correlation function (CCF) to a single exponential function: = exp(-s / (2Lp)) where theta(s) is tangent angle; s: path or contour lengths. < > denotes averaging over filaments. Results: Bundle-Lp (bundles < 15 mu m long) increased from similar to 10 to 150 mu m with increased fascin:actin ratio. The increase was similar for path-Lp (path < 15 mu m), with highly linear correlation. For longer bundle paths, the CCF-decay deviated from a single exponential, consistent with superimposition of the random path with a circular path as suggested by theoretical analysis. Conclusions: Fascin-actin bundles have similar path-Lp and bundle-Lp, both increasing with fascin:actin ratio. Path-Lp is determined by the flexural rigidity of the bundle. General significance: The findings give general insight into mechanics of cytoskeletal polymers that interact with molecular motors, aid rational development of nanotechnological applications and have implications for structure and in vivo functions of fascin-actin bundles. (C) 2014 The Authors. Published by Elsevier B.V.

Background: Bundles of unipolar actin filaments (F-actin), cross-linked via the actin-binding protein fascin, are important in filopodia of motile cells and stereocilia of inner ear sensory cells. However, such bundles are also useful as shuttles in myosin-driven nanotechnological applications. Therefore, and for elucidating aspects of biological function, we investigate if the bundle tendency to follow straight paths (quantified by path persistence length) when propelled by myosin motors is directly determined by material properties quantified by persistence length of thermally fluctuating bundles. Methods: Fluorescent bundles, labeled with rhodamine-phalloidin, were studied at fascin:actin molar ratios: 0:1 (F-actin), 1:7, 1:4 and 1:2. Persistence lengths (Lp) were obtained by fitting the cosine correlation function (CCF) to a single exponential function: = exp(-s / (2Lp)) where theta(s) is tangent angle; s: path or contour lengths. < > denotes averaging over filaments. Results: Bundle-Lp (bundles < 15 mu m long) increased from similar to 10 to 150 mu m with increased fascin:actin ratio. The increase was similar for path-Lp (path < 15 mu m), with highly linear correlation. For longer bundle paths, the CCF-decay deviated from a single exponential, consistent with superimposition of the random path with a circular path as suggested by theoretical analysis. Conclusions: Fascin-actin bundles have similar path-Lp and bundle-Lp, both increasing with fascin:actin ratio. Path-Lp is determined by the flexural rigidity of the bundle. General significance: The findings give general insight into mechanics of cytoskeletal polymers that interact with molecular motors, aid rational development of nanotechnological applications and have implications for structure and in vivo functions of fascin-actin bundles. (C) 2014 The Authors. Published by Elsevier B.V.

Background: Bundles of unipolar actin filaments (F-actin), cross-linked via the actin-binding protein fascin, are important in filopodia of motile cells and stereocilia of inner ear sensory cells. However, such bundles are also useful as shuttles in myosin-driven nanotechnological applications. Therefore, and for elucidating aspects of biological function, we investigate if the bundle tendency to follow straight paths (quantified by path persistence length) when propelled by myosin motors is directly determined by material properties quantified by persistence length of thermally fluctuating bundles. Methods: Fluorescent bundles, labeled with rhodamine-phalloidin, were studied at fascin:actin molar ratios: 0:1 (F-actin), 1:7, 1:4 and 1:2. Persistence lengths (Lp) were obtained by fitting the cosine correlation function (CCF) to a single exponential function: = exp(-s / (2Lp)) where theta(s) is tangent angle; s: path or contour lengths. < > denotes averaging over filaments. Results: Bundle-Lp (bundles < 15 mu m long) increased from similar to 10 to 150 mu m with increased fascin:actin ratio. The increase was similar for path-Lp (path < 15 mu m), with highly linear correlation. For longer bundle paths, the CCF-decay deviated from a single exponential, consistent with superimposition of the random path with a circular path as suggested by theoretical analysis. Conclusions: Fascin-actin bundles have similar path-Lp and bundle-Lp, both increasing with fascin:actin ratio. Path-Lp is determined by the flexural rigidity of the bundle. General significance: The findings give general insight into mechanics of cytoskeletal polymers that interact with molecular motors, aid rational development of nanotechnological applications and have implications for structure and in vivo functions of fascin-actin bundles. (C) 2014 The Authors. Published by Elsevier B.V.

Background: Bundles of unipolar actin filaments (F-actin), cross-linked via the actin-binding protein fascin, are important in filopodia of motile cells and stereocilia of inner ear sensory cells. However, such bundles are also useful as shuttles in myosin-driven nanotechnological applications. Therefore, and for elucidating aspects of biological function, we investigate if the bundle tendency to follow straight paths (quantified by path persistence length) when propelled by myosin motors is directly determined by material properties quantified by persistence length of thermally fluctuating bundles. Methods: Fluorescent bundles, labeled with rhodamine-phalloidin, were studied at fascin:actin molar ratios: 0:1 (F-actin), 1:7, 1:4 and 1:2. Persistence lengths (Lp) were obtained by fitting the cosine correlation function (CCF) to a single exponential function: = exp(-s / (2Lp)) where theta(s) is tangent angle; s: path or contour lengths. < > denotes averaging over filaments. Results: Bundle-Lp (bundles < 15 mu m long) increased from similar to 10 to 150 mu m with increased fascin:actin ratio. The increase was similar for path-Lp (path < 15 mu m), with highly linear correlation. For longer bundle paths, the CCF-decay deviated from a single exponential, consistent with superimposition of the random path with a circular path as suggested by theoretical analysis. Conclusions: Fascin-actin bundles have similar path-Lp and bundle-Lp, both increasing with fascin:actin ratio. Path-Lp is determined by the flexural rigidity of the bundle. General significance: The findings give general insight into mechanics of cytoskeletal polymers that interact with molecular motors, aid rational development of nanotechnological applications and have implications for structure and in vivo functions of fascin-actin bundles. (C) 2014 The Authors. Published by Elsevier B.V.

Background: Bundles of unipolar actin filaments (F-actin), cross-linked via the actin-binding protein fascin, are important in filopodia of motile cells and stereocilia of inner ear sensory cells. However, such bundles are also useful as shuttles in myosin-driven nanotechnological applications. Therefore, and for elucidating aspects of biological function, we investigate if the bundle tendency to follow straight paths (quantified by path persistence length) when propelled by myosin motors is directly determined by material properties quantified by persistence length of thermally fluctuating bundles. Methods: Fluorescent bundles, labeled with rhodamine-phalloidin, were studied at fascin:actin molar ratios: 0:1 (F-actin), 1:7, 1:4 and 1:2. Persistence lengths (Lp) were obtained by fitting the cosine correlation function (CCF) to a single exponential function: = exp(-s / (2Lp)) where theta(s) is tangent angle; s: path or contour lengths. < > denotes averaging over filaments. Results: Bundle-Lp (bundles < 15 mu m long) increased from similar to 10 to 150 mu m with increased fascin:actin ratio. The increase was similar for path-Lp (path < 15 mu m), with highly linear correlation. For longer bundle paths, the CCF-decay deviated from a single exponential, consistent with superimposition of the random path with a circular path as suggested by theoretical analysis. Conclusions: Fascin-actin bundles have similar path-Lp and bundle-Lp, both increasing with fascin:actin ratio. Path-Lp is determined by the flexural rigidity of the bundle. General significance: The findings give general insight into mechanics of cytoskeletal polymers that interact with molecular motors, aid rational development of nanotechnological applications and have implications for structure and in vivo functions of fascin-actin bundles. (C) 2014 The Authors. Published by Elsevier B.V.

A key challenge in normal development and in disease is to elucidate the mechanisms of cell migration. Here we approach this question using the tracheal system of Drosophila as a model. Tracheal cell migration requires the Breathless/FGFR pathway; however, how the pathway induces migration remains poorly understood. We find that the Breathless pathway upregulates singed at the tip of tracheal branches, and that this regulation is functionally relevant. singed encodes Drosophila Fascin, which belongs to a conserved family of actin-bundling proteins involved in cancer progression and metastasis upon misregulation. We show that singed is required for filopodia stiffness and proper morphology of tracheal tip cells, defects that correlate with an abnormal actin organisation. We propose that singed-regulated filopodia and cell fronts are required for timely and guided branch migration and for terminal branching and branch fusion. We find that singed requirements rely on its actin-bundling activity controlled by phosphorylation, and that active Singed can promote tip cell features. Furthermore, we find that singed acts in concert with forked, another actin cross-linker. The absence of both cross-linkers further stresses the relevance of tip cell morphology and filopodia for tracheal development. In summary, our results on the one hand reveal a previously undescribed role for forked in the organisation of transient actin structures such as filopodia, and on the other hand identify singed as a new target of Breathless signal, establishing a link between guidance cues, the actin cytoskeleton and tracheal morphogenesis. © 2014. Published by The Company of Biologists Ltd.

© 2014 Taylor & Francis Group, LLC. The FGFR pathway triggers a wide range of key biological responses. Among others, the Breathless (Btl, Drosophila FGFR1) receptor cascade promotes cell migration during embryonic tracheal system development. However, how the actin cytoskeleton responds to Btl pathway activation to induce cell migration has remained largely unclear. Our recent results shed light into this issue by unveiling a link between the actin-bundling protein Singed (Sn) and the Btl pathway. We showed that the Btl pathway regulates sn, which leads to the stabilization of the actin bundles required for filopodia formation and actin cytoskeleton rearrangement. This regulation contributes to tracheal migration, tracheal branch fusion and tracheal cell elongation. Parallel actin bundles (PABs) are usually cross-linked by more than one actin-bundling protein. Accordingly, we have also shown that sn synergistically interacts with forked (f), another actin crosslinker. In this Extra View we extend f analysis and hypothesize how both actin-bundling proteins may act together to regulate the PABs during tracheal embryonic development. Although both proteins are required for similar tracheal events, we suggest that Sn is essential for actin bundle initiation and stiffening, while F is required for the lengthening and further stabilization of the PABs.

© Pilar Okenve-Ramos and Marta Llimargas. Filopodia are long and thin finger-like protrusions essential for cell migration. They are formed by parallel actin bundles tightly packed by cell type and context dependent actin-bundling proteins. Our recent work analyzing the role of Fascin during tracheal development in Drosophila has shown that Singed (the Drosophila Fascin homolog) acts as a molecular link between the Branchless (FGF)/Breathless (FGFR) pathway and the actin cytoskeleton. We have reported that the lack of Singed (Sn) leads to wavy and flaccid filopodia due to the disorganization of the tracheal actin cytoskeleton. Here we describe for the first time filopodia breakage in Drosophila, and show that Fascin plays a role in this event. We propose that actin filaments in sn mutant filopodia buckle under membrane pressure due to lower bending stiffness, eventually undergoing breakage. Both Filopodia buckling and breakage would impair correct cell navigation and migration.

Filopodia are fine actin-based cellular projections used for both environmental sensing and cell motility, and they are essential organelles for metazoan cells. In this study, we reconstruct the origin of metazoan filopodia and microvilli. We first report on the evolutionary assembly of the filopodial molecular toolkit and show that homologs of many metazoan filopodial components, including fascin and myosin X, were already present in the unicellular or colonial progenitors of metazoans. Furthermore, we find that the actin crosslinking protein fascin localizes to filopodia-like structures and microvilli in the choanoflagellate Salpingoeca rosetta. In addition, homologs of filopodial genes in the holozoan Capsaspora owczarzaki are upregulated in filopodia-bearing cells relative to those that lack them. Therefore, our findings suggest that proteins essential for metazoan filopodia and microvilli are functionally conserved in unicellular and colonial holozoans and that the last common ancestor of metazoans bore a complex and specific filopodial machinery.

Filopodia are fine actin-based cellular projections used for both environmental sensing and cell motility, and they are essential organelles for metazoan cells. In this study, we reconstruct the origin of metazoan filopodia and microvilli. We first report on the evolutionary assembly of the filopodial molecular toolkit and show that homologs of many metazoan filopodial components, including fascin and myosin X, were already present in the unicellular or colonial progenitors of metazoans. Furthermore, we find that the actin crosslinking protein fascin localizes to filopodia-like structures and microvilli in the choanoflagellate Salpingoeca rosetta. In addition, homologs of filopodial genes in the holozoan Capsaspora owczarzaki are upregulated in filopodia-bearing cells relative to those that lack them. Therefore, our findings suggest that proteins essential for metazoan filopodia and microvilli are functionally conserved in unicellular and colonial holozoans and that the last common ancestor of metazoans bore a complex and specific filopodial machinery.

Filopodia are fine actin-based cellular projections used for both environmental sensing and cell motility, and they are essential organelles for metazoan cells. In this study, we reconstruct the origin of metazoan filopodia and microvilli. We first report on the evolutionary assembly of the filopodial molecular toolkit and show that homologs of many metazoan filopodial components, including fascin and myosin X, were already present in the unicellular or colonial progenitors of metazoans. Furthermore, we find that the actin crosslinking protein fascin localizes to filopodia-like structures and microvilli in the choanoflagellate Salpingoeca rosetta. In addition, homologs of filopodial genes in the holozoan Capsaspora owczarzaki are upregulated in filopodia-bearing cells relative to those that lack them. Therefore, our findings suggest that proteins essential for metazoan filopodia and microvilli are functionally conserved in unicellular and colonial holozoans and that the last common ancestor of metazoans bore a complex and specific filopodial machinery. © 2013 The Author.

Novel Roles of Actin Binding Proteins in Listeria monocytogenes Actin-Based Motility Revealed Within a Cellular ContextBy Raphael H. Buencamino Actin-based motility is essential in many biological processes from fighting harmful organisms to the development of our bodies in the correct configuration. It has been dissected to its basic parts, finding the minimal set of actin binding proteins needed to produce movement. In contrast, actin-based motility in cells allows movement at a fast pace with a diverse set of activities and structures based on the cell's immediate function within the body. We dissected the mechanism of actin-based motility in vivo and found a set of proteins whose depletion drastically changes the character and efficiency of actin based movement. We accomplished this by using Listeria monocytogenes, a pathogen that expresses one bacterial protein, ActA, that allows Listeria to hijack the host proteins necessary to move within the cell using actin-based motility. To investigate the function and role of these host actin-binding proteins in actin-based motility, in vivo, we used a form of molecular "fractionation", a powerful technique only previously available for in vitro studies. RNAi interference in the Drosophila S2 cell line provided us this form of in vivo "fractionation" by allowing selective depletion of any protein whose cellular function we wished to examine. We first examined two proteins, profilin and ADF/cofilin, whose functions have been well documented through biochemical assays and in vitro systems. We also sought to dissect actin-based motility further and determine if other actin-binding proteins are involved in its mechanism. Our hypothesis is that with over 90 actin-binding proteins implicated in actin-based motility there are other key proteins that remain uncharacterized beyond the five in the current model. Our experiments identified that CAP is an essential protein for actin-based motility in vivo. Through our investigation

Novel Roles of Actin Binding Proteins in Listeria monocytogenes Actin-Based Motility Revealed Within a Cellular ContextBy Raphael H. Buencamino Actin-based motility is essential in many biological processes from fighting harmful organisms to the development of our bodies in the correct configuration. It has been dissected to its basic parts, finding the minimal set of actin binding proteins needed to produce movement. In contrast, actin-based motility in cells allows movement at a fast pace with a diverse set of activities and structures based on the cell's immediate function within the body. We dissected the mechanism of actin-based motility in vivo and found a set of proteins whose depletion drastically changes the character and efficiency of actin based movement. We accomplished this by using Listeria monocytogenes, a pathogen that expresses one bacterial protein, ActA, that allows Listeria to hijack the host proteins necessary to move within the cell using actin-based motility. To investigate the function and role of these host actin-binding proteins in actin-based motility, in vivo, we used a form of molecular "fractionation", a powerful technique only previously available for in vitro studies. RNAi interference in the Drosophila S2 cell line provided us this form of in vivo "fractionation" by allowing selective depletion of any protein whose cellular function we wished to examine. We first examined two proteins, profilin and ADF/cofilin, whose functions have been well documented through biochemical assays and in vitro systems. We also sought to dissect actin-based motility further and determine if other actin-binding proteins are involved in its mechanism. Our hypothesis is that with over 90 actin-binding proteins implicated in actin-based motility there are other key proteins that remain uncharacterized beyond the five in the current model. Our experiments identified that CAP is an essential protein for actin-based motility in vivo. Through our investigation