Publications by authors named "Richard Firtel"

66 Publications

Phosphodiesterase PdeD, dynacortin, and a Kelch repeat-containing protein are direct GSK3 substrates in Dictyostelium that contribute to chemotaxis towards cAMP.

Environ Microbiol 2018 05;20(5):1888-1903

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0380, USA.

The migration of cells according to a diffusible chemical signal in their environment is called chemotaxis, and the slime mold Dictyostelium discoideum is widely used for the study of eukaryotic chemotaxis. Dictyostelium must sense chemicals, such as cAMP, secreted during starvation to move towards the sources of the signal. Previous work demonstrated that the gskA gene encodes the Dictyostelium homologue of glycogen synthase kinase 3 (GSK3), a highly conserved serine/threonine kinase, which plays a major role in the regulation of Dictyostelium chemotaxis. Cells lacking the GskA substrates Daydreamer and GflB exhibited chemotaxis defects less severe than those exhibited by gskA (GskA null) cells, suggesting that additional GskA substrates might be involved in chemotaxis. Using phosphoproteomics we identify the GskA substrates PdeD, dynacortin and SogA and characterize the phenotypes of their respective null cells in response to the chemoattractant cAMP. All three chemotaxis phenotypes are defective, and in addition, we determine that carboxylesterase D2 is a common downstream effector of GskA, its direct substrates PdeD, GflB and the kinases GlkA and YakA, and that it also contributes to cell migration. Our findings identify new GskA substrates in cAMP signalling and break down the essential role of GskA in myosin II regulation.
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http://dx.doi.org/10.1111/1462-2920.14126DOI Listing
May 2018

The Dictyostelium GSK3 kinase GlkA coordinates signal relay and chemotaxis in response to growth conditions.

Dev Biol 2018 03 20;435(1):56-72. Epub 2018 Jan 20.

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0380, USA. Electronic address:

GSK3 plays a central role in orchestrating key biological signaling pathways, including cell migration. Here, we identify GlkA as a GSK3 family kinase with functions that overlap with and are distinct from those of GskA. We show that GlkA, as previously shown for GskA, regulates the cell's cytoskeleton through MyoII assembly and control of Ras and Rap1 function, leading to aberrant cell migration. However, there are both qualitative and quantitative differences in the regulation of Ras and Rap1 and their downstream effectors, including PKB, PKBR1, and PI3K, with glkA cells exhibiting a more severe chemotaxis phenotype than gskA cells. Unexpectedly, the severe glkA phenotypes, but not those of gskA, are only exhibited when cells are grown attached to a substratum but not in suspension, suggesting that GlkA functions as a key kinase of cell attachment signaling. Using proteomic iTRAQ analysis we show that there are quantitative differences in the pattern of protein expression depending on the growth conditions in wild-type cells. We find that GlkA expression affects the cell's proteome during vegetative growth and development, with many of these changes depending on whether the cells are grown attached to a substratum or in suspension. These changes include key cytoskeletal and signaling proteins known to be essential for proper chemotaxis and signal relay during the aggregation stage of Dictyostelium development.
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http://dx.doi.org/10.1016/j.ydbio.2018.01.007DOI Listing
March 2018

Two-Layer Elastographic 3-D Traction Force Microscopy.

Sci Rep 2017 01 11;7:39315. Epub 2017 Jan 11.

Department of Mechanical and Aerospace Engineeing, University of California, San Diego.

Cellular traction force microscopy (TFM) requires knowledge of the mechanical properties of the substratum where the cells adhere to calculate cell-generated forces from measurements of substratum deformation. Polymer-based hydrogels are broadly used for TFM due to their linearly elastic behavior in the range of measured deformations. However, the calculated stresses, particularly their spatial patterns, can be highly sensitive to the substratum's Poisson's ratio. We present two-layer elastographic TFM (2LETFM), a method that allows for simultaneously measuring the Poisson's ratio of the substratum while also determining the cell-generated forces. The new method exploits the analytical solution of the elastostatic equation and deformation measurements from two layers of the substratum. We perform an in silico analysis of 2LETFM concluding that this technique is robust with respect to TFM experimental parameters, and remains accurate even for noisy measurement data. We also provide experimental proof of principle of 2LETFM by simultaneously measuring the stresses exerted by migrating Physarum amoeboae on the surface of polyacrylamide substrata, and the Poisson's ratio of the substrata. The 2LETFM method could be generalized to concurrently determine the mechanical properties and cell-generated forces in more physiologically relevant extracellular environments, opening new possibilities to study cell-matrix interactions.
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http://dx.doi.org/10.1038/srep39315DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5225457PMC
January 2017

Connecting G protein signaling to chemoattractant-mediated cell polarity and cytoskeletal reorganization.

Small GTPases 2018 07 14;9(4):360-364. Epub 2016 Oct 14.

a Department of Cell Biochemistry , University of Groningen , Groningen , The Netherlands.

The directional movement toward extracellular chemical gradients, a process called chemotaxis, is an important property of cells. Central to eukaryotic chemotaxis is the molecular mechanism by which chemoattractant-mediated activation of G-protein coupled receptors (GPCRs) induces symmetry breaking in the activated downstream signaling pathways. Studies with mainly Dictyostelium and mammalian neutrophils as experimental systems have shown that chemotaxis is mediated by a complex network of signaling pathways. Recently, several labs have used extensive and efficient proteomic approaches to further unravel this dynamic signaling network. Together these studies showed the critical role of the interplay between heterotrimeric G-protein subunits and monomeric G proteins in regulating cytoskeletal rearrangements during chemotaxis. Here we highlight how these proteomic studies have provided greater insight into the mechanisms by which the heterotrimeric G protein cycle is regulated, how heterotrimeric G proteins-induced symmetry breaking is mediated through small G protein signaling, and how symmetry breaking in G protein signaling subsequently induces cytoskeleton rearrangements and cell migration.
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http://dx.doi.org/10.1080/21541248.2016.1235390DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5997169PMC
July 2018

A Gα-Stimulated RapGEF Is a Receptor-Proximal Regulator of Dictyostelium Chemotaxis.

Dev Cell 2016 Jun 26;37(5):458-72. Epub 2016 May 26.

Department of Cell Biochemistry, University of Groningen, Nijenborgh 7, 9747 AG Groningen, the Netherlands. Electronic address:

Chemotaxis, or directional movement toward extracellular chemical gradients, is an important property of cells that is mediated through G-protein-coupled receptors (GPCRs). Although many chemotaxis pathways downstream of Gβγ have been identified, few Gα effectors are known. Gα effectors are of particular importance because they allow the cell to distinguish signals downstream of distinct chemoattractant GPCRs. Here we identify GflB, a Gα2 binding partner that directly couples the Dictyostelium cyclic AMP GPCR to Rap1. GflB localizes to the leading edge and functions as a Gα-stimulated, Rap1-specific guanine nucleotide exchange factor required to balance Ras and Rap signaling. The kinetics of GflB translocation are fine-tuned by GSK-3 phosphorylation. Cells lacking GflB display impaired Rap1/Ras signaling and actin and myosin dynamics, resulting in defective chemotaxis. Our observations demonstrate that GflB is an essential upstream regulator of chemoattractant-mediated cell polarity and cytoskeletal reorganization functioning to directly link Gα activation to monomeric G-protein signaling.
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http://dx.doi.org/10.1016/j.devcel.2016.05.001DOI Listing
June 2016

Cooperative cell motility during tandem locomotion of amoeboid cells.

Mol Biol Cell 2016 Apr 24;27(8):1262-71. Epub 2016 Feb 24.

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0380

Streams of migratory cells are initiated by the formation of tandem pairs of cells connected head to tail to which other cells subsequently adhere. The mechanisms regulating the transition from single to streaming cell migration remain elusive, although several molecules have been suggested to be involved. In this work, we investigate the mechanics of the locomotion ofDictyosteliumtandem pairs by analyzing the spatiotemporal evolution of their traction adhesions (TAs). We find that in migrating wild-type tandem pairs, each cell exerts traction forces on stationary sites (∼80% of the time), and the trailing cell reuses the location of the TAs of the leading cell. Both leading and trailing cells form contractile dipoles and synchronize the formation of new frontal TAs with ∼54-s time delay. Cells not expressing the lectin discoidin I or moving on discoidin I-coated substrata form fewer tandems, but the trailing cell still reuses the locations of the TAs of the leading cell, suggesting that discoidin I is not responsible for a possible chemically driven synchronization process. The migration dynamics of the tandems indicate that their TAs' reuse results from the mechanical synchronization of the leading and trailing cells' protrusions and retractions (motility cycles) aided by the cell-cell adhesions.
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http://dx.doi.org/10.1091/mbc.E15-12-0836DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4831880PMC
April 2016

Three-dimensional balance of cortical tension and axial contractility enables fast amoeboid migration.

Biophys J 2015 Feb;108(4):821-832

Department of Mechanical and Aerospace Engineering, University of California at San Diego, San Diego, California; Institute for Engineering in Medicine, University of California at San Diego, San Diego, California. Electronic address:

Fast amoeboid migration requires cells to apply mechanical forces on their surroundings via transient adhesions. However, the role these forces play in controlling cell migration speed remains largely unknown. We used three-dimensional force microscopy to measure the three-dimensional forces exerted by chemotaxing Dictyostelium cells, and examined wild-type cells as well as mutants with defects in contractility, internal F-actin crosslinking, and cortical integrity. We showed that cells pull on their substrate adhesions using two distinct, yet interconnected mechanisms: axial actomyosin contractility and cortical tension. We found that the migration speed increases when axial contractility overcomes cortical tension to produce the cell shape changes needed for locomotion. We demonstrated that the three-dimensional pulling forces generated by both mechanisms are internally balanced by an increase in cytoplasmic pressure that allows cells to push on their substrate without adhering to it, and which may be relevant for amoeboid migration in complex three-dimensional environments.
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http://dx.doi.org/10.1016/j.bpj.2014.11.3478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4336364PMC
February 2015

Cytoskeletal Mechanics Regulating Amoeboid Cell Locomotion.

Appl Mech Rev 2014 Jun;66(5)

Mechanical and Aerospace Engineering Department, Institute for Engineering in Medicine, Bioengineering Department, University of California, San Diego, La Jolla, CA.

Migrating cells exert traction forces when moving. Amoeboid cell migration is a common type of cell migration that appears in many physiological and pathological processes and is performed by a wide variety of cell types. Understanding the coupling of the biochemistry and mechanics underlying the process of migration has the potential to guide the development of pharmacological treatment or genetic manipulations to treat a wide range of diseases. The measurement of the spatiotemporal evolution of the traction forces that produce the movement is an important aspect for the characterization of the locomotion mechanics. There are several methods to calculate the traction forces exerted by the cells. Currently the most commonly used ones are traction force microscopy methods based on the measurement of the deformation induced by the cells on elastic substrate on which they are moving. Amoeboid cells migrate by implementing a motility cycle based on the sequential repetition of four phases. In this paper we review the role that specific cytoskeletal components play in the regulation of the cell migration mechanics. We investigate the role of specific cytoskeletal components regarding the ability of the cells to perform the motility cycle effectively and the generation of traction forces. The actin nucleation in the leading edge of the cell, carried by the ARP2/3 complex activated through the SCAR/WAVE complex, has shown to be fundamental to the execution of the cyclic movement and to the generation of the traction forces. The protein PIR121, a member of the SCAR/WAVE complex, is essential to the proper regulation of the periodic movement and the protein SCAR, also included in the SCAR/WAVE complex, is necessary for the generation of the traction forces during migration. The protein Myosin II, an important F-actin cross-linker and motor protein, is essential to cytoskeletal contractility and to the generation and proper organization of the traction forces during migration.
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http://dx.doi.org/10.1115/1.4026249DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4201387PMC
June 2014

Both contractile axial and lateral traction force dynamics drive amoeboid cell motility.

J Cell Biol 2014 Mar;204(6):1045-61

Department of Mechanical and Aerospace Engineering and 2 Department of Bioengineering, Jacobs School of Engineering; 3 Section of Cell and Developmental Biology, Division of Biological Sciences; and 4 Institute for Engineering in Medicine, University of California, San Diego, La Jolla, CA 92093.

Chemotaxing Dictyostelium discoideum cells adapt their morphology and migration speed in response to intrinsic and extrinsic cues. Using Fourier traction force microscopy, we measured the spatiotemporal evolution of shape and traction stresses and constructed traction tension kymographs to analyze cell motility as a function of the dynamics of the cell's mechanically active traction adhesions. We show that wild-type cells migrate in a step-wise fashion, mainly forming stationary traction adhesions along their anterior-posterior axes and exerting strong contractile axial forces. We demonstrate that lateral forces are also important for motility, especially for migration on highly adhesive substrates. Analysis of two mutant strains lacking distinct actin cross-linkers (mhcA(-) and abp120(-) cells) on normal and highly adhesive substrates supports a key role for lateral contractions in amoeboid cell motility, whereas the differences in their traction adhesion dynamics suggest that these two strains use distinct mechanisms to achieve migration. Finally, we provide evidence that the above patterns of migration may be conserved in mammalian amoeboid cells.
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http://dx.doi.org/10.1083/jcb.201307106DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3998796PMC
March 2014

Degradation of activated K-Ras orthologue via K-Ras-specific lysine residues is required for cytokinesis.

J Biol Chem 2014 Feb 13;289(7):3950-9. Epub 2013 Dec 13.

From the Division of Hematology Oncology, Department of Internal Medicine, University of Cincinnati Cancer Institute, Department of Neurosurgery, University of Cincinnati Neuroscience Institute, Brain Tumor Center University of Cincinnati, College of Medicine, Cincinnati, Ohio 45267.

Mammalian cells encode three closely related Ras proteins, H-Ras, N-Ras, and K-Ras. Oncogenic K-Ras mutations frequently occur in human cancers, which lead to dysregulated cell proliferation and genomic instability. However, mechanistic role of the Ras isoform regulation have remained largely unknown. Furthermore, the dynamics and function of negative regulation of GTP-loaded K-Ras have not been fully investigated. Here, we demonstrate RasG, the Dictyostelium orthologue of K-Ras, is targeted for degradation by polyubiquitination. Both ubiquitination and degradation of RasG were strictly associated with RasG activity. High resolution tandem mass spectrometry (LC-MS/MS) analysis indicated that RasG ubiquitination occurs at C-terminal lysines equivalent to lysines found in human K-Ras but not in H-Ras and N-Ras homologues. Substitution of these lysine residues with arginines (4KR-RasG) diminished RasG ubiquitination and increased RasG protein stability. Cells expressing 4KR-RasG failed to undergo proper cytokinesis and resulted in multinucleated cells. Ectopically expressed human K-Ras undergoes polyubiquitin-mediated degradation in Dictyostelium, whereas human H-Ras and a Dictyostelium H-Ras homologue (RasC) are refractory to ubiquitination. Our results indicate the existence of GTP-loaded K-Ras orthologue-specific degradation system in Dictyostelium, and further identification of the responsible E3-ligase may provide a novel therapeutic approach against K-Ras-mutated cancers.
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http://dx.doi.org/10.1074/jbc.M113.531178DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3924263PMC
February 2014

Three-dimensional quantification of cellular traction forces and mechanosensing of thin substrata by fourier traction force microscopy.

PLoS One 2013 4;8(9):e69850. Epub 2013 Sep 4.

Mechanical and Aerospace Engineering Department, University of California San Diego, La Jolla, California, United States of America ; Institute for Engineering in Medicine, University of California San Diego, La Jolla, California, United States of America.

We introduce a novel three-dimensional (3D) traction force microscopy (TFM) method motivated by the recent discovery that cells adhering on plane surfaces exert both in-plane and out-of-plane traction stresses. We measure the 3D deformation of the substratum on a thin layer near its surface, and input this information into an exact analytical solution of the elastic equilibrium equation. These operations are performed in the Fourier domain with high computational efficiency, allowing to obtain the 3D traction stresses from raw microscopy images virtually in real time. We also characterize the error of previous two-dimensional (2D) TFM methods that neglect the out-of-plane component of the traction stresses. This analysis reveals that, under certain combinations of experimental parameters (cell size, substratums' thickness and Poisson's ratio), the accuracy of 2D TFM methods is minimally affected by neglecting the out-of-plane component of the traction stresses. Finally, we consider the cell's mechanosensing of substratum thickness by 3D traction stresses, finding that, when cells adhere on thin substrata, their out-of-plane traction stresses can reach four times deeper into the substratum than their in-plane traction stresses. It is also found that the substratum stiffness sensed by applying out-of-plane traction stresses may be up to 10 times larger than the stiffness sensed by applying in-plane traction stresses.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0069850PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3762859PMC
April 2014

Daydreamer, a Ras effector and GSK-3 substrate, is important for directional sensing and cell motility.

Mol Biol Cell 2013 Jan 7;24(2):100-14. Epub 2012 Nov 7.

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0380, USA.

How independent signaling pathways are integrated to holistically control a biological process is not well understood. We have identified Daydreamer (DydA), a new member of the Mig10/RIAM/lamellipodin (MRL) family of adaptor proteins that localizes to the leading edge of the cell. DydA is a putative Ras effector that is required for cell polarization and directional movement during chemotaxis. dydA(-) cells exhibit elevated F-actin and assembled myosin II (MyoII), increased and extended phosphoinositide-3-kinase (PI3K) activity, and extended phosphorylation of the activation loop of PKB and PKBR1, suggesting that DydA is involved in the negative regulation of these pathways. DydA is phosphorylated by glycogen synthase kinase-3 (GSK-3), which is required for some, but not all, of DydA's functions, including the proper regulation of PKB and PKBR1 and MyoII assembly. gskA(-) cells exhibit very strong chemotactic phenotypes, as previously described, but exhibit an increased rate of random motility. gskA(-) cells have a reduced MyoII response and a reduced level of phosphatidylinositol (3,4,5)-triphosphate production, but a highly extended recruitment of PI3K to the plasma membrane and highly extended kinetics of PKB and PKBR1 activation. Our results demonstrate that GSK-3 function is essential for chemotaxis, regulating multiple substrates, and that one of these effectors, DydA, plays a key function in the dynamic regulation of chemotaxis.
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http://dx.doi.org/10.1091/mbc.E12-04-0271DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3541958PMC
January 2013

A mechanosensory system governs myosin II accumulation in dividing cells.

Mol Biol Cell 2012 Apr 29;23(8):1510-23. Epub 2012 Feb 29.

Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

The mitotic spindle is generally considered the initiator of furrow ingression. However, recent studies suggest that furrows can form without spindles, particularly during asymmetric cell division. In Dictyostelium, the mechanoenzyme myosin II and the actin cross-linker cortexillin I form a mechanosensor that responds to mechanical stress, which could account for spindle-independent contractile protein recruitment. Here we show that the regulatory and contractility network composed of myosin II, cortexillin I, IQGAP2, kinesin-6 (kif12), and inner centromeric protein (INCENP) is a mechanical stress-responsive system. Myosin II and cortexillin I form the core mechanosensor, and mechanotransduction is mediated by IQGAP2 to kif12 and INCENP. In addition, IQGAP2 is antagonized by IQGAP1 to modulate the mechanoresponsiveness of the system, suggesting a possible mechanism for discriminating between mechanical and biochemical inputs. Furthermore, IQGAP2 is important for maintaining spindle morphology and kif12 and myosin II cleavage furrow recruitment. Cortexillin II is not directly involved in myosin II mechanosensitive accumulation, but without cortexillin I, cortexillin II's role in membrane-cortex attachment is revealed. Finally, the mitotic spindle is dispensable for the system. Overall, this mechanosensory system is structured like a control system characterized by mechanochemical feedback loops that regulate myosin II localization at sites of mechanical stress and the cleavage furrow.
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http://dx.doi.org/10.1091/mbc.E11-07-0601DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3327329PMC
April 2012

Incoherent feedforward control governs adaptation of activated ras in a eukaryotic chemotaxis pathway.

Sci Signal 2012 Jan 3;5(205):ra2. Epub 2012 Jan 3.

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093, USA.

Adaptation in signaling systems, during which the output returns to a fixed baseline after a change in the input, often involves negative feedback loops and plays a crucial role in eukaryotic chemotaxis. We determined the dynamical response to a uniform change in chemoattractant concentration of a eukaryotic chemotaxis pathway immediately downstream from G protein-coupled receptors. The response of an activated Ras showed near-perfect adaptation, leading us to attempt to fit the results using mathematical models for the two possible simple network topologies that can provide perfect adaptation. Only the incoherent feedforward network accurately described the experimental results. This analysis revealed that adaptation in this Ras pathway is achieved through the proportional activation of upstream components and not through negative feedback loops. Furthermore, these results are consistent with a local excitation, global inhibition mechanism for gradient sensing, possibly with a Ras guanosine triphosphatase-activating protein acting as a global inhibitor.
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http://dx.doi.org/10.1126/scisignal.2002413DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3928814PMC
January 2012

An Oscillatory Contractile Pole-Force Component Dominates the Traction Forces Exerted by Migrating Amoeboid Cells.

Cell Mol Bioeng 2011 Dec 29;4(4):603-615. Epub 2011 Jun 29.

We used principal component analysis to dissect the mechanics of chemotaxis of amoeboid cells into a reduced set of dominant components of cellular traction forces and shape changes. The dominant traction force component in wild-type cells accounted for ~40% of the mechanical work performed by these cells, and consisted of the cell attaching at front and back contracting the substrate towards its centroid (pole-force). The time evolution of this pole-force component was responsible for the periodic variations of cell length and strain energy that the cells underwent during migration. We identified four additional canonical components, reproducible from cell to cell, overall accounting for an additional ~20% of mechanical work, and associated with events such as lateral protrusion of pseudopodia. We analyzed mutant strains with contractility defects to quantify the role that non-muscle Myosin II (MyoII) plays in amoeboid motility. In MyoII essential light chain null cells the polar-force component remained dominant. On the other hand, MyoII heavy chain null cells exhibited a different dominant traction force component, with a marked increase in lateral contractile forces, suggesting that cortical contractility and/or enhanced lateral adhesions are important for motility in this cell line. By compressing the mechanics of chemotaxing cells into a reduced set of temporally-resolved degrees of freedom, the present study may contribute to refined models of cell migration that incorporate cell-substrate interactions. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12195-011-0184-9) contains supplementary material, which is available to authorized users.
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http://dx.doi.org/10.1007/s12195-011-0184-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3234362PMC
December 2011

The SCAR/WAVE complex is necessary for proper regulation of traction stresses during amoeboid motility.

Mol Biol Cell 2011 Nov 7;22(21):3995-4003. Epub 2011 Sep 7.

Department of Bioengineering, Jacobs School of Engineering, University of California, San Diego, La Jolla, CA 92093, USA.

Cell migration requires a tightly regulated, spatiotemporal coordination of underlying biochemical pathways. Crucial to cell migration is SCAR/WAVE-mediated dendritic F-actin polymerization at the cell's leading edge. Our goal is to understand the role the SCAR/WAVE complex plays in the mechanics of amoeboid migration. To this aim, we measured and compared the traction stresses exerted by Dictyostelium cells lacking the SCAR/WAVE complex proteins PIR121 (pirA(-)) and SCAR (scrA(-)) with those of wild-type cells while they were migrating on flat, elastic substrates. We found that, compared to wild type, both mutant strains exert traction stresses of different strengths that correlate with their F-actin levels. In agreement with previous studies, we found that wild-type cells migrate by repeating a motility cycle in which the cell length and strain energy exerted by the cells on their substrate vary periodically. Our analysis also revealed that scrA(-) cells display an altered motility cycle with a longer period and a lower migration velocity, whereas pirA(-) cells migrate in a random manner without implementing a periodic cycle. We present detailed characterization of the traction-stress phenotypes of the various cell lines, providing new insights into the role of F-actin polymerization in regulating cell-substratum interactions and stresses required for motility.
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http://dx.doi.org/10.1091/mbc.E11-03-0278DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3204062PMC
November 2011

Activated membrane patches guide chemotactic cell motility.

PLoS Comput Biol 2011 Jun 30;7(6):e1002044. Epub 2011 Jun 30.

Center for Theoretical Biological Physics, University of California San Diego, La Jolla, California, United States of America.

Many eukaryotic cells are able to crawl on surfaces and guide their motility based on environmental cues. These cues are interpreted by signaling systems which couple to cell mechanics; indeed membrane protrusions in crawling cells are often accompanied by activated membrane patches, which are localized areas of increased concentration of one or more signaling components. To determine how these patches are related to cell motion, we examine the spatial localization of RasGTP in chemotaxing Dictyostelium discoideum cells under conditions where the vertical extent of the cell was restricted. Quantitative analyses of the data reveal a high degree of spatial correlation between patches of activated Ras and membrane protrusions. Based on these findings, we formulate a model for amoeboid cell motion that consists of two coupled modules. The first module utilizes a recently developed two-component reaction diffusion model that generates transient and localized areas of elevated concentration of one of the components along the membrane. The activated patches determine the location of membrane protrusions (and overall cell motion) that are computed in the second module, which also takes into account the cortical tension and the availability of protrusion resources. We show that our model is able to produce realistic amoeboid-like motion and that our numerical results are consistent with experimentally observed pseudopod dynamics. Specifically, we show that the commonly observed splitting of pseudopods can result directly from the dynamics of the signaling patches.
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http://dx.doi.org/10.1371/journal.pcbi.1002044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3127810PMC
June 2011

The LRRK2-related Roco kinase Roco2 is regulated by Rab1A and controls the actin cytoskeleton.

Mol Biol Cell 2011 Jul 5;22(13):2198-211. Epub 2011 May 5.

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0380, USA.

We identify a new pathway that is required for proper pseudopod formation. We show that Roco2, a leucine-rich repeat kinase 2 (LRRK2)-related Roco kinase, is activated in response to chemoattractant stimulation and helps mediate cell polarization and chemotaxis by regulating cortical F-actin polymerization and pseudopod extension in a pathway that requires Rab1A. We found that Roco2 binds the small GTPase Rab1A as well as the F-actin cross-linking protein filamin (actin-binding protein 120, abp120) in vivo. We show that active Rab1A (Rab1A-GTP) is required for and regulates Roco2 kinase activity in vivo and that filamin lies downstream from Roco2 and controls pseudopod extension during chemotaxis and random cell motility. Therefore our study uncovered a new signaling pathway that involves Rab1A and controls the actin cytoskeleton and pseudopod extension, and thereby, cell polarity and motility. These findings also may have implications in the regulation of other Roco kinases, including possibly LRRK2, in metazoans.
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http://dx.doi.org/10.1091/mbc.E10-12-0937DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3128523PMC
July 2011

"TORCing" neutrophil chemotaxis.

Dev Cell 2010 Dec;19(6):795-6

University of California, San Diego, La Jolla, 92093-0380, USA.

During cell migration, chemoattractant-induced signaling pathways determine the direction of movement by controlling the spatiotemporal dynamics of cytoskeletal components. In this issue of Developmental Cell, Liu et al. report that the target of rapamycin complex 2 (TORC2) controls cell polarity and chemotaxis through regulation of both F-actin and myosin II in migrating neutrophils.
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http://dx.doi.org/10.1016/j.devcel.2010.11.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3033560PMC
December 2010

A Ras signaling complex controls the RasC-TORC2 pathway and directed cell migration.

Dev Cell 2010 May;18(5):737-49

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0380, USA.

Ras was found to regulate Dictyostelium chemotaxis, but the mechanisms that spatially and temporally control Ras activity during chemotaxis remain largely unknown. We report the discovery of a Ras signaling complex that includes the Ras guanine exchange factor (RasGEF) Aimless, RasGEFH, protein phosphatase 2A (PP2A), and a scaffold designated Sca1. The Sca1/RasGEF/PP2A complex is recruited to the plasma membrane in a chemoattractant- and F-actin-dependent manner and is enriched at the leading edge of chemotaxing cells where it regulates F-actin dynamics and signal relay by controlling the activation of RasC and the downstream target of rapamycin complex 2 (TORC2)-Akt/protein kinase B (PKB) pathway. In addition, PKB and PKB-related PKBR1 phosphorylate Sca1 and regulate the membrane localization of the Sca1/RasGEF/PP2A complex, and thereby RasC activity, in a negative feedback fashion. Thus, our study uncovered a molecular mechanism whereby RasC activity and the spatiotemporal activation of TORC2 are tightly controlled at the leading edge of chemotaxing cells.
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http://dx.doi.org/10.1016/j.devcel.2010.03.017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2893887PMC
May 2010

Involvement of the cytoskeleton in controlling leading-edge function during chemotaxis.

Mol Biol Cell 2010 Jun 7;21(11):1810-24. Epub 2010 Apr 7.

Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0380, USA.

In response to directional stimulation by a chemoattractant, cells rapidly activate a series of signaling pathways at the site closest to the chemoattractant source that leads to F-actin polymerization, pseudopod formation, and directional movement up the gradient. Ras proteins are major regulators of chemotaxis in Dictyostelium; they are activated at the leading edge, are required for chemoattractant-mediated activation of PI3K and TORC2, and are one of the most rapid responders, with activity peaking at approximately 3 s after stimulation. We demonstrate that in myosin II (MyoII) null cells, Ras activation is highly extended and is not restricted to the site closest to the chemoattractant source. This causes elevated, extended, and spatially misregulated activation of PI3K and TORC2 and their effectors Akt/PKB and PKBR1, as well as elevated F-actin polymerization. We further demonstrate that disruption of specific IQGAP/cortexillin complexes, which also regulate cortical mechanics, causes extended activation of PI3K and Akt/PKB but not Ras activation. Our findings suggest that MyoII and IQGAP/cortexillin play key roles in spatially and temporally regulating leading-edge activity and, through this, the ability of cells to restrict the site of pseudopod formation.
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http://dx.doi.org/10.1091/mbc.e10-01-0009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2877640PMC
June 2010

Myosin II is essential for the spatiotemporal organization of traction forces during cell motility.

Mol Biol Cell 2010 Feb 2;21(3):405-17. Epub 2009 Dec 2.

Section of Cell and Developmental Biology, Division of Biological Sciences, Department of Mechanical and Aerospace Engineering, and Department of Bioengineering, University of California, San Diego, La Jolla, CA 92093, USA.

Amoeboid motility requires spatiotemporal coordination of biochemical pathways regulating force generation and consists of the quasi-periodic repetition of a motility cycle driven by actin polymerization and actomyosin contraction. Using new analytical tools and statistical methods, we provide, for the first time, a statistically significant quantification of the spatial distribution of the traction forces generated at each phase of the cycle (protrusion, contraction, retraction, and relaxation). We show that cells are constantly under tensional stress and that wild-type cells develop two opposing "pole" forces pulling the front and back toward the center whose strength is modulated up and down periodically in each cycle. We demonstrate that nonmuscular myosin II complex (MyoII) cross-linking and motor functions have different roles in controlling the spatiotemporal distribution of traction forces, the changes in cell shape, and the duration of all the phases. We show that the time required to complete each phase is dramatically increased in cells with altered MyoII motor function, demonstrating that it is required not only for contraction but also for protrusion. Concomitant loss of MyoII actin cross-linking leads to a force redistribution throughout the cell perimeter pulling inward toward the center. However, it does not reduce significantly the magnitude of the traction forces, uncovering a non-MyoII-mediated mechanism for the contractility of the cell.
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http://dx.doi.org/10.1091/mbc.e09-08-0703DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2814786PMC
February 2010

Spatiotemporal regulation of Ras-GTPases during chemotaxis.

Methods Mol Biol 2009 ;571:333-48

Department of Systems Biology, Harvard Medical School and Division of Signal Transduction, Beth Israel Deaconess Medical Center, Boston, MA, USA.

Many eukaryotic cells can elicit intracellular signaling relays to produce pseudopodia and move up to the chemoattractant gradient (chemotaxis) or move randomly in the absence of extracellular stimuli and nutrients (random movement). A precise spatiotemporal regulation of Ras-GTPases, such as Ras and Rap, is crucial to induce pseudopodia formation and cellular adhesion during the chemotaxis and random movement. Here, we describe biochemical and real-time imaging methods for using Dictyostelium to understand the signaling events important for chemotaxis and random cell movement. The chapter includes (1) a biochemical method to assess Ras and Rap1 activation in response to chemoattractant, (2) an imaging method to detect endogenous Ras and Rap1 activation in moving cells, and (3) a simultaneous imaging method to decipher the precise order and localization of these signaling events. With a combination of powerful Dictyostelium genetics, these methods will facilitate to elucidate a dynamic activation of Ras proteins and their inter relay with other signaling molecules during chemotaxis and random movement.
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http://dx.doi.org/10.1007/978-1-60761-198-1_23DOI Listing
January 2010

Mechanosensing through cooperative interactions between myosin II and the actin crosslinker cortexillin I.

Curr Biol 2009 Sep 30;19(17):1421-8. Epub 2009 Jul 30.

Department of Cell Biology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Background: Mechanosensing governs many processes from molecular to organismal levels, including during cytokinesis where it ensures successful and symmetrical cell division. Although many proteins are now known to be force sensitive, myosin motors with their ATPase activity and force-sensitive mechanical steps are well poised to facilitate cellular mechanosensing. For a myosin motor to experience tension, the actin filament must also be anchored.

Results: Here, we find a cooperative relationship between myosin II and the actin crosslinker cortexillin I where both proteins are essential for cellular mechanosensory responses. Although many functions of cortexillin I and myosin II are dispensable for cytokinesis, all are required for full mechanosensing. Our analysis demonstrates that this mechanosensor has three critical elements: the myosin motor where the lever arm acts as a force amplifier, a force-sensitive bipolar thick-filament assembly, and a long-lived actin crosslinker, which anchors the actin filament so that the motor may experience tension. We also demonstrate that a Rac small GTPase inhibits this mechanosensory module during interphase, allowing the module to be primarily active during cytokinesis.

Conclusions: Overall, myosin II and cortexillin I define a cellular-scale mechanosensor that controls cell shape during cytokinesis. This system is exquisitely tuned through the enzymatic properties of the myosin motor, its lever arm length, and bipolar thick-filament assembly dynamics. The system also requires cortexillin I to stably anchor the actin filament so that the myosin motor can experience tension. Through this cross-talk, myosin II and cortexillin I define a cellular-scale mechanosensor that monitors and corrects shape defects, ensuring symmetrical cell division.
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http://dx.doi.org/10.1016/j.cub.2009.07.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2763054PMC
September 2009

Regulation of Dictyostelium morphogenesis by RapGAP3.

Dev Biol 2009 Apr 22;328(2):210-20. Epub 2009 Jan 22.

Section of Cell and Developmental Biology, Division of Biological Sciences, Center for Molecular Genetics, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0380, USA.

Rap1 is a key regulator of cell adhesion and cell motility in Dictyostelium. Here, we identify a Rap1-specific GAP protein (RapGAP3) and provide evidence that Rap1 signaling regulates cell-cell adhesion and cell migration within the multicellular organism. RapGAP3 mediates the deactivation of Rap1 at the late mound stage of development and plays an important role in regulating cell sorting during apical tip formation, when the anterior-posterior axis of the organism is formed, by controlling cell-cell adhesion and cell migration. The loss of RapGAP3 results in a severely altered morphogenesis of the multicellular organism at the late mound stage. Direct measurement of cell motility within the mound shows that rapGAP3(-) cells have a reduced speed of movement and, compared to wild-type cells, have a reduced motility towards the apex. rapGAP3(-) cells exhibit some increased EDTA/EGTA sensitive cell-cell adhesion at the late mound stage. RapGAP3 transiently and rapidly translocates to the cell cortex in response to chemoattractant stimulation, which is dependent on F-actin polymerization. We suggest that the altered morphogenesis and the cell-sorting defect of rapGAP3(-) cells may result in reduced directional movement of the mutant cells to the apex of the mound.
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http://dx.doi.org/10.1016/j.ydbio.2009.01.016DOI Listing
April 2009

Dictyostelium Dock180-related RacGEFs regulate the actin cytoskeleton during cell motility.

Mol Biol Cell 2009 Jan 26;20(2):699-707. Epub 2008 Nov 26.

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California, San Diego, La Jolla, CA 92093-0380, USA.

Cell motility of amoeboid cells is mediated by localized F-actin polymerization that drives the extension of membrane protrusions to promote forward movements. We show that deletion of either of two members of the Dictyostelium Dock180 family of RacGEFs, DockA and DockD, causes decreased speed of chemotaxing cells. The phenotype is enhanced in the double mutant and expression of DockA or DockD complements the reduced speed of randomly moving DockD null cells' phenotype, suggesting that DockA and DockD are likely to act redundantly and to have similar functions in regulating cell movement. In this regard, we find that overexpressing DockD causes increased cell speed by enhancing F-actin polymerization at the sites of pseudopod extension. DockD localizes to the cell cortex upon chemoattractant stimulation and at the leading edge of migrating cells and this localization is dependent on PI3K activity, suggesting that DockD might be part of the pathway that links PtdIns(3,4,5)P(3) production to F-actin polymerization. Using a proteomic approach, we found that DdELMO1 is associated with DockD and that Rac1A and RacC are possible in vivo DockD substrates. In conclusion, our work provides a further understanding of how cell motility is controlled and provides evidence that the molecular mechanism underlying Dock180-related protein function is evolutionarily conserved.
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http://dx.doi.org/10.1091/mbc.e08-09-0899DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2626555PMC
January 2009

Spatiotemporal regulation of Ras activity provides directional sensing.

Curr Biol 2008 Oct;18(20):1587-1593

Section of Cell and Developmental Biology Division of Biological Sciences Center for Molecular Genetics University of California, San Diego 9500 Gilman Drive La Jolla, CA 92093-0380 USA.

Cells' ability to detect and orient themselves in chemoattractant gradients has been the subject of numerous studies, but the underlying molecular mechanisms remain largely unknown [1]. Ras activation is the earliest polarized response to chemoattractant gradients downstream from heterotrimeric G proteins in Dictyostelium, and inhibition of Ras signaling results in directional migration defects [2]. Activated Ras is enriched at the leading edge, promoting the localized activation of key chemotactic effectors, such as PI3K and TORC2 [2-5]. To investigate the role of Ras in directional sensing, we studied the effect of its misregulation by using cells with disrupted RasGAP activity. We identified an ortholog of mammalian NF1, DdNF1, as a major regulator of Ras activity in Dictyostelium. We show that disruption of nfaA leads to spatially and temporally unregulated Ras activity, causing cytokinesis and chemotaxis defects. By using unpolarized, latrunculin-treated cells, we show that tight regulation of Ras is important for gradient sensing. Together, our findings suggest that Ras is part of the cell's compass and that the RasGAP-mediated regulation of Ras activity affects directional sensing.
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http://dx.doi.org/10.1016/j.cub.2008.08.069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2590931PMC
October 2008

Regulation of contractile vacuole formation and activity in Dictyostelium.

EMBO J 2008 Aug 17;27(15):2064-76. Epub 2008 Jul 17.

Section of Cell and Developmental Biology, Division of Biological Sciences, University of California San Diego, La Jolla, CA 92093-0380, USA.

The contractile vacuole (CV) system is the osmoregulatory organelle required for survival for many free-living cells under hypotonic conditions. We identified a new CV regulator, Disgorgin, a TBC-domain-containing protein, which translocates to the CV membrane at the late stage of CV charging and regulates CV-plasma membrane fusion and discharging. disgorgin(-) cells produce large CVs due to impaired CV-plasma membrane fusion. Disgorgin is a specific GAP for Rab8A-GTP, which also localizes to the CV and whose hydrolysis is required for discharging. We demonstrate that Drainin, a previously identified TBC-domain-containing protein, lies upstream from Disgorgin in this pathway. Unlike Disgorgin, Drainin lacks GAP activity but functions as a Rab11A effector. The BEACH family proteins LvsA and LvsD were identified in a suppressor/enhancer screen of the disgorgin(-) large CV phenotype and demonstrated to have distinct functions in regulating CV formation. Our studies help define the pathways controlling CV function.
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http://dx.doi.org/10.1038/emboj.2008.131DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2516880PMC
August 2008

Directional sensing during chemotaxis.

FEBS Lett 2008 Jun 29;582(14):2075-85. Epub 2008 Apr 29.

Department of Biological Sciences, Vanderbilt University, Nashville, TN 37235, USA.

Cells have the innate ability to sense and move towards a variety of chemoattractants. We investigate the pathways by which cells sense and respond to chemoattractant gradients. We focus on the model system Dictyostelium and compare our understanding of chemotaxis in this system with recent advances made using neutrophils and other mammalian cell types, which share many molecular components and signaling pathways with Dictyostelium. This review also examines models that have been proposed to explain how cells are able to respond to small differences in ligand concentrations between the anterior leading edge and posterior of the cell. In addition, we highlight the overlapping functions of many signaling components in diverse processes beyond chemotaxis, including random cell motility and cell division.
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http://dx.doi.org/10.1016/j.febslet.2008.04.035DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2519798PMC
June 2008
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