Publications by authors named "Louis Hodgson"

61 Publications

Metalloprotease ADAMTS-1 decreases cell migration and invasion modulating the spatiotemporal dynamics of Cdc42 activity.

Cell Signal 2021 Jan 6;77:109827. Epub 2020 Nov 6.

Department of Cell and Developmental Biology, Institute of Biomedical Sciences, University of Sao Paulo, Av. Prof. Lineu Prestes 1524, Ed Biomédicas 1 sala 428, São Paulo, SP 05508-000, Brazil. Electronic address:

ADAMTSs (A Disintegrin And Metalloproteinase with ThromboSpondin motifs) are secreted proteases dependent on Zn/Ca, involved in physiological and pathological processes and are part of the extracellular matrix (ECM). Here, we investigated if ADAMTS-1 is required for invasion and migration of cells and the possible mechanism involved. In order to test ADAMTS-1's role in ovarian cancer cells (CHO, NIH-OVCAR-3 and ES2) and NIH-3 T3 fibroblasts, we modified the levels of ADAMTS-1 and compared those to parental. Cells exposed to ADAMTS-1-enriched medium exhibited a decline in cell migration and invasion when compared to controls with or without a functional metalloproteinase domain. The opposite was observed in cells when ADAMTS-1 was deleted via the CRISPR/Cas9 approach. The decline in ADAMTS-1 levels enhanced the phosphorylated form of Src and FAK. We also evaluated the activities of cellular Rho GTPases from cell lysates using the GLISA® kit. The Cdc42-GTP signal was significantly increased in the CRISPR ADAMTS-1 ES-2 cells. By a Förster resonance energy transfer (FRET) biosensor for Cdc42 activity in ES-2 cells we demonstrated that Cdc42 activity was strongly polarized at the leading edge of migrating cells with ADAMTS-1 deletion, compared to the wild type cells. As conclusion, ADAMTS-1 inhibits proliferation, polarization and migration.
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http://dx.doi.org/10.1016/j.cellsig.2020.109827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7723338PMC
January 2021

Novel phospho-switch function of delta-catenin in dendrite development.

J Cell Biol 2020 11;219(11)

Department of Genetics, The University of Texas MD Anderson Cancer Center, Houston, TX.

In neurons, dendrites form the major sites of information receipt and integration. It is thus vital that, during development, the dendritic arbor is adequately formed to enable proper neural circuit formation and function. While several known processes shape the arbor, little is known of those that govern dendrite branching versus extension. Here, we report a new mechanism instructing dendrites to branch versus extend. In it, glutamate signaling activates mGluR5 receptors to promote Ckd5-mediated phosphorylation of the C-terminal PDZ-binding motif of delta-catenin. The phosphorylation state of this motif determines delta-catenin's ability to bind either Pdlim5 or Magi1. Whereas the delta:Pdlim5 complex enhances dendrite branching at the expense of elongation, the delta:Magi1 complex instead promotes lengthening. Our data suggest that these complexes affect dendrite development by differentially regulating the small-GTPase RhoA and actin-associated protein Cortactin. We thus reveal a "phospho-switch" within delta-catenin, subject to a glutamate-mediated signaling pathway, that assists in balancing the branching versus extension of dendrites during neural development.
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http://dx.doi.org/10.1083/jcb.201909166DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534926PMC
November 2020

Differential regulation of rho GTPases during lung adenocarcinoma migration and invasion reveals a novel role of the tumor suppressor StarD13 in invadopodia regulation.

Cell Commun Signal 2020 09 8;18(1):144. Epub 2020 Sep 8.

Department of Natural Sciences, School of Arts and Sciences, Lebanese American University, P.O. Box: 13-5053. Chouran, Beirut, 1102 2801, Lebanon.

Background: Lung cancer is the second most commonly occurring cancer. The ability to metastasize and spread to distant locations renders the tumor more aggressive. Members of the Rho subfamily of small GTP-binding proteins (GTPases) play a central role in the regulation of the actin cytoskeleton and in cancer cell migration and metastasis. In this study we investigated the role of the RhoA/Cdc42 GAP, StarD13, a previously described tumor suppressor, in malignancy, migration and invasion of the lung cancer cells A549.

Methods: We knocked down StarD13 expression in A549 lung cancer cells and tested the effect on cell migration and invadopodia formation using time lapse imaging and invasion assays. We also performed rescue experiments to determine the signaling pathways downstream of StarD13 and transfected the cells with FRET biosensors for RhoGTPases to identify the proteins involved in invadopodia formation.

Results: We observed a decrease in the level of expression of StarD13 in lung tumor tissues compared to normal lung tissues through immunohistochemistry. StarD13 also showed a lower expression in the lung adenocarcinoma cell line A549 compared to normal lung cells, WI38. In addition, the depletion of StarD13 increased cell proliferation and viability in WI38 and A549 cells, suggesting that StarD13 might potentially be a tumor suppressor in lung cancer. The depletion of StarD13, however, inhibited cell motility, conversely demonstrating a positive regulatory role in cell migration. This was potentially due to the constitutive activation of RhoA detected by pull down and FRET assays. Surprisingly, StarD13 suppressed cell invasion by inhibiting Cdc42-mediated invadopodia formation. Indeed, TKS4 staining and invadopodia assay revealed that StarD13 depletion increased Cdc42 activation as well as invadopodia formation and matrix degradation. Normal lung cells depleted of StarD13 also produced invadopodia, otherwise a unique hallmark of invasive cancer cells. Cdc42 knock down mimicked the effects of StarD13, while overexpression of a constitutively active Cdc42 mimicked the effects of its depletion. Finally, immunostaining and FRET analysis revealed the absence of StarD13 in invadopodia as compared to Cdc42, which was activated in invadopodia at the sites of matrix degradation.

Conclusion: In conclusion, StarD13 plays distinct roles in lung cancer cell migration and invasion through its differential regulation of Rho GTPases. Video abstract.
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http://dx.doi.org/10.1186/s12964-020-00635-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7487901PMC
September 2020

Optogenetic regulation of endogenous proteins.

Nat Commun 2020 01 30;11(1):605. Epub 2020 Jan 30.

Medicum, Faculty of Medicine, University of Helsinki, Helsinki, 00290, Finland.

Techniques of protein regulation, such as conditional gene expression, RNA interference, knock-in and knock-out, lack sufficient spatiotemporal accuracy, while optogenetic tools suffer from non-physiological response due to overexpression artifacts. Here we present a near-infrared light-activatable optogenetic system, which combines the specificity and orthogonality of intrabodies with the spatiotemporal precision of optogenetics. We engineer optically-controlled intrabodies to regulate genomically expressed protein targets and validate the possibility to further multiplex protein regulation via dual-wavelength optogenetic control. We apply this system to regulate cytoskeletal and enzymatic functions of two non-tagged endogenous proteins, actin and RAS GTPase, involved in complex functional networks sensitive to perturbations. The optogenetically-enhanced intrabodies allow fast and reversible regulation of both proteins, as well as simultaneous monitoring of RAS signaling with visible-light biosensors, enabling all-optical approach. Growing number of intrabodies should make their incorporation into optogenetic tools the versatile technology to regulate endogenous targets.
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http://dx.doi.org/10.1038/s41467-020-14460-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6992714PMC
January 2020

Optogenetics: Rho GTPases Activated by Light in Living Macrophages.

Methods Mol Biol 2020 ;2108:281-293

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA.

Genetically encoded optogenetic tools are increasingly popular and useful for perturbing signaling pathways with high spatial and temporal resolution in living cells. Here, we show basic procedures employed to implement optogenetics of Rho GTPases in a macrophage cell line. Methods described here are generally applicable to other genetically encoded optogenetic tools utilizing the blue-green spectrum of light for activation, designed for specific proteins and enzymatic targets important for immune cell functions.
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http://dx.doi.org/10.1007/978-1-0716-0247-8_24DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7032845PMC
January 2021

Kalirin/Trio Rho GDP/GTP exchange factors regulate proinsulin and insulin secretion.

J Mol Endocrinol 2018 Nov 1. Epub 2018 Nov 1.

R Kuliawat, Developmental and Molecular Biology, Albert Einstein College of Medicine, Bronx, 10461, United States.

Key features for progression to pancreatic β-cell failure and disease are loss of glucose responsiveness and an increased ratio of secreted proinsulin to insulin. Proinsulin and insulin are stored in secretory granules (SGs) and the fine-tuning of hormone output requires signal mediated recruitment of select SG populations according to intracellular location and age. The GTPase Rac1 coordinates multiple signaling pathways that specify SG release and Rac1 activity is controlled in part by GDP/GTP exchange factors (GEFs). To explore the function of two large multidomain GEFs, Kalirin and Trio in β-cells, we manipulated their Rac1-specific GEF1 domain activity by using small molecule inhibitors and by genetically ablating Kalirin. We examined age related secretory granule behavior employing radiolabeling protocols. Loss of Kalirin/Trio function attenuated radioactive proinsulin release by reducing constitutive-like secretion and exocytosis of 2-hour old granules. At later chase times or at steady state, Kalirin/Trio manipulations decreased glucose stimulated insulin output. Finally, use of a Rac1 FRET biosensor with cultured β-cell lines, demonstrated that Kalirin/Trio GEF1 activity was required for normal rearrangement of Rac1 to the plasma membrane in response to glucose. Rac1 activation can be evoked by both glucose metabolism and signaling through the incretin glucagon-like peptide 1 (GLP-1) receptor. GLP-1 addition restored Rac1 localization/activity and insulin secretion in the absence of Kalirin, thereby assigning Kalirin's participation to stimulatory glucose signaling.
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http://dx.doi.org/10.1530/JME-18-0048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6494717PMC
November 2018

Matrix mechanical plasticity regulates cancer cell migration through confining microenvironments.

Nat Commun 2018 10 8;9(1):4144. Epub 2018 Oct 8.

Department of Mechanical Engineering, Stanford University, Stanford, CA, 94305, USA.

Studies of cancer cell migration have found two modes: one that is protease-independent, requiring micron-sized pores or channels for cells to squeeze through, and one that is protease-dependent, relevant for confining nanoporous matrices such as basement membranes (BMs). However, many extracellular matrices exhibit viscoelasticity and mechanical plasticity, irreversibly deforming in response to force, so that pore size may be malleable. Here we report the impact of matrix plasticity on migration. We develop nanoporous and BM ligand-presenting interpenetrating network (IPN) hydrogels in which plasticity could be modulated independent of stiffness. Strikingly, cells in high plasticity IPNs carry out protease-independent migration through the IPNs. Mechanistically, cells in high plasticity IPNs extend invadopodia protrusions to mechanically and plastically open up micron-sized channels and then migrate through them. These findings uncover a new mode of protease-independent migration, in which cells can migrate through confining matrix if it exhibits sufficient mechanical plasticity.
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http://dx.doi.org/10.1038/s41467-018-06641-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6175826PMC
October 2018

Macropinosome formation by tent pole ruffling in macrophages.

J Cell Biol 2018 11 27;217(11):3873-3885. Epub 2018 Aug 27.

Institute for Molecular Bioscience (IMB) and IMB Centre for Inflammation and Disease Research, The University of Queensland, Brisbane, Australia

Pathogen-mediated activation of macrophages arms innate immune responses that include enhanced surface ruffling and macropinocytosis for environmental sampling and receptor internalization and signaling. Activation of macrophages with bacterial lipopolysaccharide (LPS) generates prominent dorsal ruffles, which are precursors for macropinosomes. Very rapid, high-resolution imaging of live macrophages with lattice light sheet microscopy (LLSM) reveals new features and actions of dorsal ruffles, which redefine the process of macropinosome formation and closure. We offer a new model in which ruffles are erected and supported by F-actin tent poles that cross over and twist to constrict the forming macropinosomes. This process allows for formation of large macropinosomes induced by LPS. We further describe the enrichment of active Rab13 on tent pole ruffles and show that CRISPR deletion of Rab13 results in aberrant tent pole ruffles and blocks the formation of large LPS-induced macropinosomes. Based on the exquisite temporal and spatial resolution of LLSM, we can redefine the ruffling and macropinosome processes that underpin innate immune responses.
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http://dx.doi.org/10.1083/jcb.201804137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6219714PMC
November 2018

Characterization of Genetically Encoded FRET Biosensors for Rho-Family GTPases.

Methods Mol Biol 2018 ;1821:87-106

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA.

Genetically encoded FRET-based biosensors are increasingly popular and useful tools for examining signaling pathways with high spatial and temporal resolution in living cells. Here, we show basic techniques used to characterize and to validate single-chain, genetically encoded Förster resonance energy transfer (FRET) biosensors of the Rho GTPase-family proteins. Methods described here are generally applicable to other genetically encoded FRET-based biosensors by modifying the tested conditions to include additional/different regulators and inhibitors, as appropriate for the specific protein of interest.
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http://dx.doi.org/10.1007/978-1-4939-8612-5_7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6104821PMC
March 2019

Publisher Correction: Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET.

Nat Chem Biol 2018 Sep;14(9):902

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA.

In the version of this article originally published, the values for time shown on the x axis of Figure 5c were incorrect. The error has been corrected in all versions of the paper.
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http://dx.doi.org/10.1038/s41589-018-0070-zDOI Listing
September 2018

Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET.

Nat Chem Biol 2018 06 23;14(6):591-600. Epub 2018 Apr 23.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY, USA.

Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Förster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.
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http://dx.doi.org/10.1038/s41589-018-0044-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5964015PMC
June 2018

Asymmetric localization of DLC1 defines avian trunk neural crest polarity for directional delamination and migration.

Nat Commun 2017 10 30;8(1):1185. Epub 2017 Oct 30.

School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Hong Kong, China.

Following epithelial-mesenchymal transition, acquisition of avian trunk neural crest cell (NCC) polarity is prerequisite for directional delamination and migration, which in turn is essential for peripheral nervous system development. However, how this cell polarization is established and regulated remains unknown. Here we demonstrate that, using the RHOA biosensor in vivo and in vitro, the initiation of NCC polarization is accompanied by highly activated RHOA in the cytoplasm at the cell rear and its fluctuating activity at the front edge. This differential RHOA activity determines polarized NC morphology and motility, and is regulated by the asymmetrically localized RhoGAP Deleted in liver cancer (DLC1) in the cytoplasm at the cell front. Importantly, the association of DLC1 with NEDD9 is crucial for its asymmetric localization and differential RHOA activity. Moreover, NC specifiers, SOX9 and SOX10, regulate NEDD9 and DLC1 expression, respectively. These results present a SOX9/SOX10-NEDD9/DLC1-RHOA regulatory axis to govern NCC migratory polarization.
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http://dx.doi.org/10.1038/s41467-017-01107-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5662599PMC
October 2017

Rac3 regulates breast cancer invasion and metastasis by controlling adhesion and matrix degradation.

J Cell Biol 2017 12 23;216(12):4331-4349. Epub 2017 Oct 23.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY

The initial step of metastasis is the local invasion of tumor cells into the surrounding tissue. Invadopodia are actin-based protrusions that mediate the matrix degradation necessary for invasion and metastasis of tumor cells. We demonstrate that Rac3 GTPase is critical for integrating the adhesion of invadopodia to the extracellular matrix (ECM) with their ability to degrade the ECM in breast tumor cells. We identify two pathways at invadopodia important for integrin activation and delivery of matrix metalloproteinases: through the upstream recruiter CIB1 as well as the downstream effector GIT1. Rac3 activity, at and surrounding invadopodia, is controlled by Vav2 and βPIX. These guanine nucleotide exchange factors regulate the spatiotemporal dynamics of Rac3 activity, impacting GIT1 localization. Moreover, the GTPase-activating function of GIT1 toward the vesicular trafficking regulator Arf6 GTPase is required for matrix degradation. Importantly, Rac3 regulates the ability of tumor cells to metastasize in vivo. The Rac3-dependent mechanisms we show in this study are critical for balancing proteolytic activity and adhesive activity to achieve a maximally invasive phenotype.
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http://dx.doi.org/10.1083/jcb.201704048DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5716284PMC
December 2017

The Role of Rho-GTPases and actin polymerization during Macrophage Tunneling Nanotube Biogenesis.

Sci Rep 2017 08 17;7(1):8547. Epub 2017 Aug 17.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave, Gruss MRRC 306, Bronx, NY, 10461, USA.

Macrophage interactions with other cells, either locally or at distances, are imperative in both normal and pathological conditions. While soluble means of communication can transmit signals between different cells, it does not account for all long distance macrophage interactions. Recently described tunneling nanotubes (TNTs) are membranous channels that connect cells together and allow for transfer of signals, vesicles, and organelles. However, very little is known about the mechanism by which these structures are formed. Here we investigated the signaling pathways involved in TNT formation by macrophages using multiple imaging techniques including super-resolution microscopy (3D-SIM) and live-cell imaging including the use of FRET-based Rho GTPase biosensors. We found that formation of TNTs required the activity and differential localization of Cdc42 and Rac1. The downstream Rho GTPase effectors mediating actin polymerization through Arp2/3 nucleation, Wiskott-Aldrich syndrome protein (WASP) and WASP family verprolin-homologous 2 (WAVE2) proteins are also important, and both pathways act together during TNT biogenesis. Finally, TNT function as measured by transfer of cellular material between cells was reduced following depletion of a single factor demonstrating the importance of these factors in TNTs. Given that the characterization of TNT formation is still unclear in the field; this study provides new insights and would enhance the understanding of TNT formation towards investigating new markers.
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http://dx.doi.org/10.1038/s41598-017-08950-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5561213PMC
August 2017

Mesenchymal Cell Invasion Requires Cooperative Regulation of Persistent Microtubule Growth by SLAIN2 and CLASP1.

Dev Cell 2016 Dec 8;39(6):708-723. Epub 2016 Dec 8.

Cell Biology, Department of Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands.

Microtubules regulate signaling, trafficking, and cell mechanics, but the respective contribution of these functions to cell morphogenesis and migration in 3D matrices is unclear. Here, we report that the microtubule plus-end tracking protein (+TIP) SLAIN2, which suppresses catastrophes, is not required for 2D cell migration but is essential for mesenchymal cell invasion in 3D culture and in a mouse cancer model. We show that SLAIN2 inactivation does not affect Rho GTPase activity, trafficking, and focal adhesion formation. However, SLAIN2-dependent catastrophe inhibition determines microtubule resistance to compression and pseudopod elongation. Another +TIP, CLASP1, is also needed to form invasive pseudopods because it prevents catastrophes specifically at their tips. When microtubule growth persistence is reduced, inhibition of depolymerization is sufficient for pseudopod maintenance but not remodeling. We propose that catastrophe inhibition by SLAIN2 and CLASP1 supports mesenchymal cell shape in soft 3D matrices by enabling microtubules to perform a load-bearing function.
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http://dx.doi.org/10.1016/j.devcel.2016.11.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5178967PMC
December 2016

Macrophage-dependent tumor cell transendothelial migration is mediated by Notch1/Mena-initiated invadopodium formation.

Sci Rep 2016 11 30;6:37874. Epub 2016 Nov 30.

Department of Anatomy and Structural Biology Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461, United States.

The process of intravasation involving transendothelial migration is a key step in metastatic spread. How the triple cell complex composed of a macrophage, Mena over-expressing tumor cell and endothelial cell, called the tumor microenvironment of metastasis (TMEM), facilitates tumor cell transendothelial migration is not completely understood. Previous work has shown that the physical contact between a macrophage and tumor cell results in the formation of invadopodia, actin-rich matrix degrading protrusions, important for tumor cell invasion and transendothelial migration and tumor cell dissemination. Herein, we show that the macrophage-induced invadopodium is formed through a Notch1/Mena signaling pathway in the tumor cell upon macrophage contact. This heterotypic tumor cell - macrophage interaction results in the upregulation of Mena through the activation of MENA transcription. Notch1 and Mena expression are required for tumor cell transendothelial migration, a necessary step during intravasation. Inhibition of the Notch signaling pathway blocked macrophage-induced invadopodium formation in vitro and the dissemination of tumor cells from the primary tumor in vivo. Our findings indicate a novel role for Notch1 signaling in the regulation of Mena expression and transendothelial migration and provide mechanistic information essential to the use of therapeutic inhibitors of metastasis.
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http://dx.doi.org/10.1038/srep37874DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5129016PMC
November 2016

Control of mitochondrial function and cell growth by the atypical cadherin Fat1.

Nature 2016 11 9;539(7630):575-578. Epub 2016 Nov 9.

Wilf Family Cardiovascular Research Institute, Department of Medicine (Cardiology), Albert Einstein College of Medicine, Bronx, New York 10461, USA.

Mitochondrial products such as ATP, reactive oxygen species, and aspartate are key regulators of cellular metabolism and growth. Abnormal mitochondrial function compromises integrated growth-related processes such as development and tissue repair, as well as homeostatic mechanisms that counteract ageing and neurodegeneration, cardiovascular disease, and cancer. Physiologic mechanisms that control mitochondrial activity in such settings remain incompletely understood. Here we show that the atypical Fat1 cadherin acts as a molecular 'brake' on mitochondrial respiration that regulates vascular smooth muscle cell (SMC) proliferation after arterial injury. Fragments of Fat1 accumulate in SMC mitochondria, and the Fat1 intracellular domain interacts with multiple mitochondrial proteins, including critical factors associated with the inner mitochondrial membrane. SMCs lacking Fat1 (Fat1) grow faster, consume more oxygen for ATP production, and contain more aspartate. Notably, expression in Fat1 cells of a modified Fat1 intracellular domain that localizes exclusively to mitochondria largely normalizes oxygen consumption, and the growth advantage of these cells can be suppressed by inhibition of mitochondrial respiration, which suggest that a Fat1-mediated growth control mechanism is intrinsic to mitochondria. Consistent with this idea, Fat1 species associate with multiple respiratory complexes, and Fat1 deletion both increases the activity of complexes I and II and promotes the formation of complex-I-containing supercomplexes. In vivo, Fat1 is expressed in injured human and mouse arteries, and inactivation of SMC Fat1 in mice potentiates the response to vascular damage, with markedly increased medial hyperplasia and neointimal growth, and evidence of higher SMC mitochondrial respiration. These studies suggest that Fat1 controls mitochondrial activity to restrain cell growth during the reparative, proliferative state induced by vascular injury. Given recent reports linking Fat1 to cancer, abnormal kidney and muscle development, and neuropsychiatric disease, this Fat1 function may have importance in other settings of altered cell growth and metabolism.
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http://dx.doi.org/10.1038/nature20170DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5257202PMC
November 2016

Using Fluorescence Resonance Energy Transfer-Based Biosensors to Probe Rho GTPase Activation During Phagocytosis.

Methods Mol Biol 2017 ;1519:125-143

Departments of Anatomy and Structural Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY, 10461, USA.

The p21-family members of Rho GTPases are important for the control of actin cytoskeleton dynamics, and are critical regulators of phagocytosis. The three-dimensional structure of phagosomes and the highly compartmentalized nature of the signaling mechanisms during phagocytosis require high-resolution imaging using ratiometric biosensors to decipher Rho GTPase activities regulating phagosome formation and function. Here we describe methods for the expression and ratiometric imaging of FRET-based Rho GTPase biosensors in macrophages during phagocytosis. As an example, we show Cdc42 activity at the phagosome over Z-serial planes. In addition, we demonstrate the usage of a new, fast, and user-friendly deconvolution package that delivers significant improvements in the attainable details of Rho GTPase activity in phagosome structures.
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http://dx.doi.org/10.1007/978-1-4939-6581-6_9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5116239PMC
January 2018

Correcting mitochondrial fusion by manipulating mitofusin conformations.

Nature 2016 12 24;540(7631):74-79. Epub 2016 Oct 24.

Center for Pharmacogenomics, Department of Internal Medicine, Washington University School of Medicine, St. Louis, Missouri, USA.

Mitochondria are dynamic organelles that exchange contents and undergo remodelling during cyclic fusion and fission. Genetic mutations in MFN2 (the gene encoding mitofusin 2) interrupt mitochondrial fusion and cause the untreatable neurodegenerative condition Charcot-Marie-Tooth disease type 2A (CMT2A). It has not yet been possible to directly modulate mitochondrial fusion, in part because the structural basis of mitofusin function is not completely understood. Here we show that mitofusins adopt either a fusion-constrained or a fusion-permissive molecular conformation, directed by specific intramolecular binding interactions, and demonstrate that mitofusin-dependent mitochondrial fusion can be regulated in mouse cells by targeting these conformational transitions. On the basis of this model, we engineered a cell-permeant minipeptide to destabilize the fusion-constrained conformation of mitofusin and promote the fusion-permissive conformation, reversing mitochondrial abnormalities in cultured fibroblasts and neurons that harbour CMT2A-associated genetic defects. The relationship between the conformational plasticity of mitofusin 2 and mitochondrial dynamism reveals a central mechanism that regulates mitochondrial fusion, the manipulation of which can correct mitochondrial pathology triggered by defective or imbalanced mitochondrial dynamics.
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http://dx.doi.org/10.1038/nature20156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5315023PMC
December 2016

FRET binding antenna reports spatiotemporal dynamics of GDI-Cdc42 GTPase interactions.

Nat Chem Biol 2016 10 8;12(10):802-809. Epub 2016 Aug 8.

Department of Pharmacology and Lineberger Cancer Center, University of North Carolina at Chapel Hill.

Guanine-nucleotide dissociation inhibitors (GDIs) are negative regulators of Rho family GTPases that sequester the GTPases away from the membrane. Here we ask how GDI-Cdc42 interaction regulates localized Cdc42 activation for cell motility. The sensitivity of cells to overexpression of Rho family pathway components led us to a new biosensor, GDI.Cdc42 FLARE, in which Cdc42 is modified with a fluorescence resonance energy transfer (FRET) 'binding antenna' that selectively reports Cdc42 binding to endogenous GDIs. Similar antennae could also report GDI-Rac1 and GDI-RhoA interaction. Through computational multiplexing and simultaneous imaging, we determined the spatiotemporal dynamics of GDI-Cdc42 interaction and Cdc42 activation during cell protrusion and retraction. This revealed remarkably tight coordination of GTPase release and activation on a time scale of 10 s, suggesting that GDI-Cdc42 interactions are a critical component of the spatiotemporal regulation of Cdc42 activity, and not merely a mechanism for global sequestration of an inactivated pool of signaling molecules.
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http://dx.doi.org/10.1038/nchembio.2145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5030135PMC
October 2016

Optical Tools To Study the Isoform-Specific Roles of Small GTPases in Immune Cells.

J Immunol 2016 Apr 7;196(8):3479-93. Epub 2016 Mar 7.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, NY 10461; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, NY 10461

Despite the 92% homology of the hematopoietic cell-specific Rac2 to the canonical isoform Rac1, these isoforms have been shown to play nonredundant roles in immune cells. To study isoform-specific dynamics of Rac in live cells, we developed a genetically encoded, single-chain FRET-based biosensor for Rac2. We also made significant improvements to our existing single-chain Rac1 biosensor. We optimized the biosensor constructs for facile expression in hematopoietic cells and performed functional validations in murine macrophage sublines of RAW264.7 cells. Rac2, Rac1, and Cdc42 have been implicated in the formation of actin-rich protrusions by macrophages, but their individual activation dynamics have not been previously characterized. We found that both Rac1 and Rac2 had similar activation kinetics, yet they had distinct spatial distributions in response to the exogenous stimulus, fMLF. Active Rac1 was mainly localized to the cell periphery, whereas active Rac2 was distributed throughout the cell, with an apparent higher concentration in the perinuclear region. We also performed an extensive morphodynamic analysis of Rac1, Rac2, and Cdc42 activities during the extension of random protrusions. We found that Rac2 appears to play a leading role in the generation of random protrusions, as we observed an initial strong activation of Rac2 in regions distal from the leading edge, followed by the activation of Rac1, a second burst of Rac2 and then Cdc42 immediately behind the leading edge. Overall, isoform-specific biosensors that have been optimized for expression should be valuable for interrogating the coordination of Rho family GTPase activities in living cells.
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http://dx.doi.org/10.4049/jimmunol.1501655DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4821714PMC
April 2016

Synonymous modification results in high-fidelity gene expression of repetitive protein and nucleotide sequences.

Genes Dev 2015 Apr 15;29(8):876-86. Epub 2015 Apr 15.

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA; Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA

Repetitive nucleotide or amino acid sequences are often engineered into probes and biosensors to achieve functional readouts and robust signal amplification. However, these repeated sequences are notoriously prone to aberrant deletion and degradation, impacting the ability to correctly detect and interpret biological functions. Here, we introduce a facile and generalizable approach to solve this often unappreciated problem by modifying the nucleotide sequences of the target mRNA to make them nonrepetitive but still functional ("synonymous"). We first demonstrated the procedure by designing a cassette of synonymous MS2 RNA motifs and tandem coat proteins for RNA imaging and showed a dramatic improvement in signal and reproducibility in single-RNA detection in live cells. The same approach was extended to enhancing the stability of engineered fluorescent biosensors containing a fluorescent resonance energy transfer (FRET) pair of fluorescent proteins on which a great majority of systems thus far in the field are based. Using the synonymous modification to FRET biosensors, we achieved correct expression of full-length sensors, eliminating the aberrant truncation products that often were assumed to be due to nonspecific proteolytic cleavages. Importantly, the biological interpretations of the sensor are significantly different when a correct, full-length biosensor is expressed. Thus, we show here a useful and generally applicable method to maintain the integrity of expressed genes, critical for the correct interpretation of probe readouts.
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http://dx.doi.org/10.1101/gad.259358.115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4403262PMC
April 2015

DENND2B activates Rab13 at the leading edge of migrating cells and promotes metastatic behavior.

J Cell Biol 2015 Mar 23;208(5):629-48. Epub 2015 Feb 23.

Department of Neurology and Neurosurgery, Montreal Neurological Institute; and Department of Biochemistry, Goodman Cancer Centre; McGill University, Montreal, Quebec H3A 0G4, Canada

The small guanosine triphosphatase Rab13 functions in exocytic vesicle trafficking in epithelial cells. Alterations in Rab13 activity have been observed in human cancers, yet the mechanism of Rab13 activation and its role in cancer progression remain unclear. In this paper, we identify the DENN domain protein DENND2B as the guanine nucleotide exchange factor for Rab13 and develop a novel Förster resonance energy transfer-based Rab biosensor to reveal activation of Rab13 by DENND2B at the leading edge of migrating cells. DENND2B interacts with the Rab13 effector MICAL-L2 at the cell periphery, and this interaction is required for the dynamic remodeling of the cell's leading edge. Disruption of Rab13-mediated trafficking dramatically limits the invasive behavior of epithelial cells in vitro and the growth and migration of highly invasive cancer cells in vivo. Thus, blocking Rab13 activation by DENND2B may provide a novel target to limit the spread of epithelial cancers.
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http://dx.doi.org/10.1083/jcb.201407068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4347646PMC
March 2015

Forword. Regulation of RhoGTPases in motility: A fine balancing act.

Authors:
Louis Hodgson

Cell Adh Migr 2014 ;8(6):525

a Department of Anatomy and Structural Biology; Gruss-Lipper Biophotonics Center; Albert Einstein Cancer Center ; Albert Einstein College of Medicine of Yeshiva University ; Bronx , NY USA.

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http://dx.doi.org/10.1080/19336918.2014.1000242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594230PMC
July 2015

Rho GTPase isoforms in cell motility: Don't fret, we have FRET.

Cell Adh Migr 2014 ;8(6):526-34

a Department of Anatomy and Structural Biology ; Albert Einstein College of Medicine of Yeshiva University ; Bronx , NY USA.

The Rho-family of p21 small GTPases are directly linked to the regulation of actin-based motile machinery and play a key role in the control of cell migration. Aside from the original and most well-characterized canonical Rho GTPases RhoA, Rac1, and Cdc42, numerous isoforms of these key proteins have been identified and shown to have specific roles in regulating various cellular motility processes. The major difficulty in addressing these isoform-specific effects is that isoforms typically contain highly similar primary amino acid sequences and thus are able to interact with the same upstream regulators and the downstream effector targets. Here, we will introduce the major members of each GTPase subfamily and discuss recent advances in the design and application of fluorescent resonance energy transfer-based probes, which are at the forefront of the technologies available to directly probe the differential, spatiotemporal activation dynamics of these proteins in live single cells. Currently, it is possible to specifically detect the activation status of RhoA vs. RhoC isoforms, as well as Cdc42 vs. TC-10 isoforms in living cells. Clearly, additional efforts are still required to produce biosensor systems capable of detecting other isoforms of Rho GTPases including RhoB, Rac2/3, RhoG, etc. Through such efforts, we will uncover the isoform-specific roles of these near-identical proteins in living cells, clearly an important area of the Rho GTPase biology that is not yet fully appreciated.
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http://dx.doi.org/10.4161/cam.29712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4594258PMC
July 2015

Induction of entosis by epithelial cadherin expression.

Cell Res 2014 Nov 24;24(11):1288-98. Epub 2014 Oct 24.

1] Cell Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA [2] BCMB Allied Program, Weill Cornell Medical College, 1300 York Avenue, New York, NY 10065, USA.

Cell engulfment typically targets dead or dying cells for clearance from metazoan tissues. However, recent evidence demonstrates that live cells can also be targeted and that engulfment can cause cell death. Entosis is one mechanism proposed to mediate the engulfment and killing of live tumor cells by their neighbors, an activity often referred to as cell cannibalism. Here we report that the expression of exogenous epithelial cadherin proteins (E- or P-cadherin) in human breast tumor cells lacking endogenous expression of epithelial cadherins induces entosis and inhibits transformed growth. Entosis induced by cadherin expression is associated with the polarized distribution of Rho and Rho-kinase (ROCK) activity within entotic cells, which is dependent on p190A RhoGAP activity. ROCK inhibition or downregulation of p190A RhoGAP expression reduces entosis and increases the transformed growth of epithelial cadherin-expressing tumor cells. These data define new cell systems for the study of entosis, and identify entosis as a mechanism of cell cannibalism that is induced by the establishment of epithelial adhesion and inhibits transformed growth.
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http://dx.doi.org/10.1038/cr.2014.137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4220160PMC
November 2014

Western analysis of intracellular interleukin-8 in human mononuclear leukocytes.

Methods Mol Biol 2014 ;1172:285-93

Department of Anatomy and Structural Biology, Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Avenue, Bronx, NY, 10461, USA,

Most cytokines are stored in the cytoplasm until their release into the extracellular environment; however, some cytokines have been reported to localize in the nucleus. Traditional whole cell extract preparation does not provide information about the intracellular localization of cytokines. Here, we describe how to prepare cytoplasmic and nuclear extracts that can be analyzed by immunoblotting. While in this chapter we use this method to analyze intracellular localization of interleukin-8 (IL-8) in human mononuclear leukocytes, this protocol is adaptable to any cell type or protein of interest.
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http://dx.doi.org/10.1007/978-1-4939-0928-5_26DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4163682PMC
January 2015

A mix-and-measure assay for determining the activation status of endogenous Cdc42 in cytokine-stimulated macrophage cell lysates.

Methods Mol Biol 2014 ;1172:173-84

Department of Anatomy and Structural Biology, Albert Einstein College of Medicine of Yeshiva University, 1300 Morris Park Ave, Bronx, NY, 10461, USA,

Cytokine stimulations of leukocytes many times result in transient activation of the p21 Rho family of small GTPases. The role of these molecules during cell migration and chemotaxis is well established. The traditional approach to study the activation dynamics of these proteins involves affinity pull-downs that are often cumbersome and prone to errors. Here, we describe a reagent and a method of simple "mix-and-measure" approach useful for determining the activation status of endogenous Cdc42 GTPase from cell lysates.
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http://dx.doi.org/10.1007/978-1-4939-0928-5_15DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4133094PMC
January 2015

Talin regulates moesin-NHE-1 recruitment to invadopodia and promotes mammary tumor metastasis.

J Cell Biol 2014 Jun 2;205(5):737-51. Epub 2014 Jun 2.

Department of Anatomy and Structural Biology and Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461Department of Anatomy and Structural Biology and Gruss Lipper Biophotonics Center, Albert Einstein College of Medicine of Yeshiva University, Bronx, NY 10461

Invadopodia are actin-rich protrusions that degrade the extracellular matrix and are required for stromal invasion, intravasation, and metastasis. The role of the focal adhesion protein talin in regulating these structures is not known. Here, we demonstrate that talin is required for invadopodial matrix degradation and three-dimensional extracellular matrix invasion in metastatic breast cancer cells. The sodium/hydrogen exchanger 1 (NHE-1) is linked to the cytoskeleton by ezrin/radixin/moesin family proteins and is known to regulate invadopodium-mediated matrix degradation. We show that the talin C terminus binds directly to the moesin band 4.1 ERM (FERM) domain to recruit a moesin-NHE-1 complex to invadopodia. Silencing talin resulted in a decrease in cytosolic pH at invadopodia and blocked cofilin-dependent actin polymerization, leading to impaired invadopodium stability and matrix degradation. Furthermore, talin is required for mammary tumor cell motility, intravasation, and spontaneous lung metastasis in vivo. Thus, our findings provide a novel understanding of how intracellular pH is regulated and a molecular mechanism by which talin enhances tumor cell invasion and metastasis.
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http://dx.doi.org/10.1083/jcb.201312046DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4050723PMC
June 2014