scholarly journals Essential and nonredundant roles for Diaphanous formins in cortical microtubule capture and directed cell migration

2014 ◽  
Vol 25 (5) ◽  
pp. 658-668 ◽  
Author(s):  
Pascale Daou ◽  
Salma Hasan ◽  
Dennis Breitsprecher ◽  
Emilie Baudelet ◽  
Luc Camoin ◽  
...  
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Affinity Purification ◽  
Cortical Microtubule ◽  
Leading Edge ◽  
Large Family ◽  
Mass Spectrometry Analysis ◽  
Cell Cortex ◽  
Spectrometry Analysis ◽  
Microtubule Capture

Formins constitute a large family of proteins that regulate the dynamics and organization of both the actin and microtubule cytoskeletons. Previously we showed that the formin mDia1 helps tether microtubules at the cell cortex, acting downstream of the ErbB2 receptor tyrosine kinase. Here we further study the contributions of mDia1 and its two most closely related formins, mDia2 and mDia3, to cortical microtubule capture and ErbB2-dependent breast carcinoma cell migration. We find that depletion of each of these three formins strongly disrupts chemotaxis without significantly affecting actin-based structures. Further, all three formins are required for formation of cortical microtubules in a nonredundant manner, and formin proteins defective in actin polymerization remain active for microtubule capture. Using affinity purification and mass spectrometry analysis, we identify differential binding partners of the formin-homology domain 2 (FH2) of mDia1, mDia2, and mDia3, which may explain their nonredundant roles in microtubule capture. The FH2 domain of mDia1 specifically interacts with Rab6-interacting protein 2 (Rab6IP2). Further, mDia1 is required for cortical localization of Rab6IP2, and concomitant depletion of Rab6IP2 and IQGAP1 severely disrupts cortical capture of microtubules, demonstrating the coinvolvement of mDia1, IQGAP1, and Rab6IP2 in microtubule tethering at the leading edge.

scholarly journals Cortical dynamics during cell motility are regulated by CRL3KLHL21 E3 ubiquitin ligase

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Thibault Courtheoux ◽  
Radoslav I. Enchev ◽  
Fabienne Lampert ◽  
Juan Gerez ◽  
Jochen Beck ◽  
...  
Keyword(s):  
Cell Migration ◽  
Ubiquitin Ligase ◽  
Cortical Plasticity ◽  
Cortical Microtubule ◽  
E3 Ubiquitin Ligase ◽  
Cell Movement ◽  
Focal Adhesions ◽  
Leading Edge ◽  
Cell Cortex ◽  
Cortical Dynamics

Abstract Directed cell movement involves spatial and temporal regulation of the cortical microtubule (Mt) and actin networks to allow focal adhesions (FAs) to assemble at the cell front and disassemble at the rear. Mts are known to associate with FAs, but the mechanisms coordinating their dynamic interactions remain unknown. Here we show that the CRL3KLHL21 E3 ubiquitin ligase promotes cell migration by controlling Mt and FA dynamics at the cell cortex. Indeed, KLHL21 localizes to FA structures preferentially at the leading edge, and in complex with Cul3, ubiquitylates EB1 within its microtubule-interacting CH-domain. Cells lacking CRL3KLHL21 activity or expressing a non-ubiquitylatable EB1 mutant protein are unable to migrate and exhibit strong defects in FA dynamics, lamellipodia formation and cortical plasticity. Our study thus reveals an important mechanism to regulate cortical dynamics during cell migration that involves ubiquitylation of EB1 at focal adhesions.

scholarly journals A reverse signaling pathway downstream of Sema4A controls cell migration via Scrib

2016 ◽  
Vol 216 (1) ◽  
pp. 199-215 ◽  
Author(s):  
Tianliang Sun ◽  
Lida Yang ◽  
Harmandeep Kaur ◽  
Jenny Pestel ◽  
Mario Looso ◽  
...  
Keyword(s):  
Cell Migration ◽  
Signaling Pathway ◽  
Large Family ◽  
Mass Spectrometry Analysis ◽  
Cellular Functions ◽  
Spectrometry Analysis ◽  
Reverse Signaling ◽  
Downstream Effector ◽  
Guanosine Triphosphatase ◽  
Interfering Rna

Semaphorins comprise a large family of ligands that regulate key cellular functions through their receptors, plexins. In this study, we show that the transmembrane semaphorin 4A (Sema4A) can also function as a receptor, rather than a ligand, and transduce signals triggered by the binding of Plexin-B1 through reverse signaling. Functionally, reverse Sema4A signaling regulates the migration of various cancer cells as well as dendritic cells. By combining mass spectrometry analysis with small interfering RNA screening, we identify the polarity protein Scrib as a downstream effector of Sema4A. We further show that binding of Plexin-B1 to Sema4A promotes the interaction of Sema4A with Scrib, thereby removing Scrib from its complex with the Rac/Cdc42 exchange factor βPIX and decreasing the activity of the small guanosine triphosphatase Rac1 and Cdc42. Our data unravel a role for Plexin-B1 as a ligand and Sema4A as a receptor and characterize a reverse signaling pathway downstream of Sema4A, which controls cell migration.

scholarly journals Contribution of Filopodia to Cell Migration: A Mechanical Link between Protrusion and Contraction

2010 ◽  
Vol 2010 ◽  
pp. 1-13 ◽  
Author(s):  
Fei Xue ◽  
Deanna M. Janzen ◽  
David A. Knecht
Keyword(s):  
Cell Migration ◽  
Actin Polymerization ◽  
Myosin Ii ◽  
Temporal Distribution ◽  
Leading Edge ◽  
Distal Portion ◽  
Actin Binding ◽  
Actin Networks ◽  
Motile Cells ◽  
A Cell

Numerous F-actin containing structures are involved in regulating protrusion of membrane at the leading edge of motile cells. We have investigated the structure and dynamics of filopodia as they relate to events at the leading edge and the function of the trailing actin networks. We have found that although filopodia contain parallel bundles of actin, they contain a surprisingly nonuniform spatial and temporal distribution of actin binding proteins. Along the length of the actin filaments in a single filopodium, the most distal portion contains primarily T-plastin, while the proximal portion is primarily bound byα-actinin and coronin. Some filopodia are stationary, but lateral filopodia move with respect to the leading edge. They appear to form a mechanical link between the actin polymerization network at the front of the cell and the myosin motor activity in the cell body. The direction of lateral filopodial movement is associated with the direction of cell migration. When lateral filopodia initiate from and move toward only one side of a cell, the cell will turn opposite to the direction of filopodial flow. Therefore, this filopodia-myosin II system allows actin polymerization driven protrusion forces and myosin II mediated contractile force to be mechanically coordinated.

scholarly journals Identification of sulfenylated cysteines in Arabidopsis thaliana proteins using a disulfide-linked peptide reporter

2020 ◽  
Author(s):  
Bo Wei ◽  
Patrick Willems ◽  
Jingjing Huang ◽  
Caiping Tian ◽  
Jing Yang ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Protein Interactions ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Technological Advancement ◽  
Amino Acid Residues ◽  
Cysteine Oxidation ◽  
Spectrometry Analysis ◽  
Mixed Disulfide ◽  
And Function

ABSTRACTIn proteins, hydrogen peroxide (H2O2) reacts with redox-sensitive cysteines to form cysteine sulfenic acid, also known as S-sulfenylation. These cysteine oxidation events can steer diverse cellular processes by altering protein interactions, trafficking, conformation, and function. Previously, we had identified S-sulfenylated proteins by using a tagged proteinaceous probe based on the yeast AP-1–like (Yap1) transcription factor that specifically reacts with sulfenic acids and traps them through a mixed disulfide bond. However, the identity of the S-sulfenylated amino acid residues remained enigmatic. Here, we present a technological advancement to identify in situ sulfenylated cysteines directly by means of the transgenic Yap1 probe. In Arabidopsis thaliana cells, after an initial affinity purification and a tryptic digestion, we further enriched the mixed disulfide-linked peptides with an antibody targeting the YAP1C-derived peptide (C598SEIWDR) that entails the redox-active cysteine. Subsequent mass spectrometry analysis with pLink 2 identified 1,745 YAP1C cross-linked peptides, indicating sulfenylated cysteines in over 1,000 proteins. Approximately 55% of these YAP1C-linked cysteines had previously been reported as redox-sensitive cysteines (S-sulfenylation, S-nitrosylation, and reversibly oxidized cysteines). The presented methodology provides a noninvasive approach to identify sulfenylated cysteines in any species that can be genetically modified.

scholarly journals Subgenomic flavivirus RNA binds the mosquito DEAD/H-box helicase ME31B and determines Zika virus transmission by Aedes aegypti

2019 ◽  
Vol 116 (38) ◽  
pp. 19136-19144 ◽  
Author(s):  
Giel P. Göertz ◽  
Joyce W. M. van Bree ◽  
Anwar Hiralal ◽  
Bas M. Fernhout ◽  
Carmen Steffens ◽  
...  
Keyword(s):  
Aedes Aegypti ◽  
Zika Virus ◽  
Affinity Purification ◽  
Mechanistic Model ◽  
Virus Transmission ◽  
Protein Translation ◽  
Mass Spectrometry Analysis ◽  
Small Interfering Rnas ◽  
Spectrometry Analysis ◽  
Mosquito Infection

Zika virus (ZIKV) is an arthropod-borne flavivirus predominantly transmitted by Aedes aegypti mosquitoes and poses a global human health threat. All flaviviruses, including those that exclusively replicate in mosquitoes, produce a highly abundant, noncoding subgenomic flavivirus RNA (sfRNA) in infected cells, which implies an important function of sfRNA during mosquito infection. Currently, the role of sfRNA in flavivirus transmission by mosquitoes is not well understood. Here, we demonstrate that an sfRNA-deficient ZIKV (ZIKVΔSF1) replicates similar to wild-type ZIKV in mosquito cell culture but is severely attenuated in transmission by Ae. aegypti after an infectious blood meal, with 5% saliva-positive mosquitoes for ZIKVΔSF1 vs. 31% for ZIKV. Furthermore, viral titers in the mosquito saliva were lower for ZIKVΔSF1 as compared to ZIKV. Comparison of mosquito infection via infectious blood meals and intrathoracic injections showed that sfRNA is important for ZIKV to overcome the mosquito midgut barrier and to promote virus accumulation in the saliva. Next-generation sequencing of infected mosquitoes showed that viral small-interfering RNAs were elevated upon ZIKVΔSF1 as compared to ZIKV infection. RNA-affinity purification followed by mass spectrometry analysis uncovered that sfRNA specifically interacts with a specific set of Ae. aegypti proteins that are normally associated with RNA turnover and protein translation. The DEAD/H-box helicase ME31B showed the highest affinity for sfRNA and displayed antiviral activity against ZIKV in Ae. aegypti cells. Based on these results, we present a mechanistic model in which sfRNA sequesters ME31B to promote flavivirus replication and virion production to facilitate transmission by mosquitoes.

scholarly journals ProbingN-glycoprotein microheterogeneity by lectin affinity purification-mass spectrometry analysis

2019 ◽  
Vol 10 (19) ◽  
pp. 5146-5155 ◽  
Author(s):  
Di Wu ◽  
Jingwen Li ◽  
Weston B. Struwe ◽  
Carol V. Robinson
Keyword(s):  
Mass Spectrometry ◽  
Protein Level ◽  
Affinity Purification ◽  
Mass Spectrometry Analysis ◽  
Intact Protein ◽  
Spectrometry Analysis ◽  
Lectin Affinity ◽  
Affinity Purification Mass Spectrometry

A lectin affinity purification-mass spectrometry approach to characterize lectin-reactive glycoproteoforms and elucidate lectin specificities at the intact protein level.

scholarly journals Identification of Novel Surface Proteins of Anaplasma phagocytophilum by Affinity Purification and Proteomics

2007 ◽  
Vol 189 (21) ◽  
pp. 7819-7828 ◽  
Author(s):  
Yan Ge ◽  
Yasuko Rikihisa
Keyword(s):  
Anaplasma Phagocytophilum ◽  
Affinity Purification ◽  
The United States ◽  
Capillary Liquid Chromatography ◽  
Etiologic Agent ◽  
Surface Proteins ◽  
Mass Spectrometry Analysis ◽  
Spectrometry Analysis ◽  
Major Surface

ABSTRACT Anaplasma phagocytophilum is the etiologic agent of human granulocytic anaplasmosis (HGA), one of the major tick-borne zoonoses in the United States. The surface of A. phagocytophilum plays a crucial role in subverting the hostile host cell environment. However, except for the P44/Msp2 outer membrane protein family, the surface components of A. phagocytophilum are largely unknown. To identify the major surface proteins of A. phagocytophilum, a membrane-impermeable, cleavable biotin reagent, sulfosuccinimidyl-2-[biotinamido]ethyl-1,3-dithiopropionate (Sulfo-NHS-SS-Biotin), was used to label intact bacteria. The biotinylated bacterial surface proteins were isolated by streptavidin agarose affinity purification and then separated by electrophoresis, followed by capillary liquid chromatography-nanospray tandem mass spectrometry analysis. Among the major proteins captured by affinity purification were five A. phagocytophilum proteins, Omp85, hypothetical proteins APH_0404 (designated Asp62) and APH_0405 (designated Asp55), P44 family proteins, and Omp-1A. The surface exposure of Asp62 and Asp55 was verified by immunofluorescence microscopy. Recombinant Asp62 and Asp55 proteins were recognized by an HGA patient serum. Anti-Asp62 and anti-Asp55 peptide sera partially neutralized A. phagocytophilum infection of HL-60 cells in vitro. We found that the Asp62 and Asp55 genes were cotranscribed and conserved among members of the family Anaplasmataceae. With the exception of P44-18, all of the proteins were newly revealed major surface-exposed proteins whose study should facilitate understanding the interaction between A. phagocytophilum and the host. These proteins may serve as targets for development of chemotherapy, diagnostics, and vaccines.

scholarly journals Hsp110 is required for spindle length control

2012 ◽  
Vol 198 (4) ◽  
pp. 623-636 ◽  
Author(s):  
Taras Makhnevych ◽  
Philip Wong ◽  
Oxana Pogoutse ◽  
Franco J. Vizeacoumar ◽  
Jack F. Greenblatt ◽  
...  
Keyword(s):  
Affinity Purification ◽  
Spindle Assembly ◽  
S Phase ◽  
Mass Spectrometry Analysis ◽  
Nucleotide Exchange ◽  
Spectrometry Analysis ◽  
Nucleotide Exchange Factor ◽  
Spindle Elongation ◽  
Chaperone Complex

Systematic affinity purification combined with mass spectrometry analysis of N- and C-tagged cytoplasmic Hsp70/Hsp110 chaperones was used to identify new roles of Hsp70/Hsp110 in the cell. This allowed the mapping of a chaperone–protein network consisting of 1,227 unique interactions between the 9 chaperones and 473 proteins and highlighted roles for Hsp70/Hsp110 in 14 broad biological processes. Using this information, we uncovered an essential role for Hsp110 in spindle assembly and, more specifically, in modulating the activity of the widely conserved kinesin-5 motor Cin8. The role of Hsp110 Sse1 as a nucleotide exchange factor for the Hsp70 chaperones Ssa1/Ssa2 was found to be required for maintaining the proper distribution of kinesin-5 motors within the spindle, which was subsequently required for bipolar spindle assembly in S phase. These data suggest a model whereby the Hsp70–Hsp110 chaperone complex antagonizes Cin8 plus-end motility and prevents premature spindle elongation in S phase.

scholarly journals Involvement of Cell Surface HSP90 in Cell Migration Reveals a Novel Role in the Developing Nervous System

2004 ◽  
Vol 279 (44) ◽  
pp. 45379-45388 ◽  
Author(s):  
Katerina Sidera ◽  
Martina Samiotaki ◽  
Eleni Yfanti ◽  
George Panayotou ◽  
Evangelia Patsavoudi
Keyword(s):  
Nervous System ◽  
Cell Migration ◽  
Heat Shock ◽  
Cell Surface ◽  
Antigen Expression ◽  
Mass Spectrometry Analysis ◽  
Rhodamine Phalloidin ◽  
Spectrometry Analysis ◽  
Hsp90 Activity ◽  
Surface Localization

Heat shock protein HSP90 plays important roles in cellular regulation, primarily as a chaperone for a number of key intracellular proteins. We report here that the two HSP90 isoforms, α and β, also localize on the surface of cells in the nervous system and are involved in their migration. A 94-kDa surface antigen, the 4C5 antigen, which was previously shown to be involved in migration processes during development of the nervous system, is shown to be identical to HSP90α using mass spectrometry analysis. This identity is further confirmed by immunoprecipitation experiments and by induction of 4C5 antigen expression in heat shock-treated embryonic rat brain cultures. Moreover, immunocytochemistry on live cerebellar rat cells reveals cell surface localization of both HSP90α and -β. Cell migration from cerebellar and sciatic nerve explants is inhibited by anti-HSP90α and anti-HSP90β antibodies, similarly to the inhibition observed with monoclonal antibody 4C5. Moreover, immunostaining with rhodamine-phalloidin of migrating Schwann cells cultured in the presence of antibodies against both α and β isoforms of HSP90 reveals that HSP90 activity is associated with actin cytoskeletal organization, necessary for lamellipodia formation.

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