Association between α4 integrin cytoplasmic tail and non-muscle myosin IIA regulates cell migration.

α4ß1 integrin regulates cell migration via cytoplasmic interactions. Here, we report an association between the cytoplasmic tail of α4 integrin (α4 tail) and non-muscle myosin IIA (MIIA), demonstrated by co-immunoprecipitation of the MIIA heavy chain (HC) with anti-α4-integrin antibodies and pull-down of MIIA-HC with recombinant α4 tail from cell lysates. The association between the α4 tail and MIIA does not require paxillin binding or phosphorylation at Ser988 in the α4 tail. We found that substituting Glu982 in the α4 tail with alanine (E982A) disrupts the α4-MIIA association without interfering with the paxillin binding or Ser988 phosphorylation. By comparing stably transfected CHO cells, we show that the E982A mutation reduces the ability of α4ß1 integrin to mediate cell spreading and to promote front-back polarization. In addition, we show that E982A impairs shear-flow-induced migration of the α4-integrin-expressing CHO cells by reducing their migration speed and directional persistence. The E982A mutation also leads to defects in the organization of MIIA filament bundles. Furthermore, when cells are plated on fibronectin and simulated with shear flow, α4ß1 integrin forms filament-like patterns that co-align with MIIA filament bundles. These results provide a new mechanism for linking integrins to the actomyosin cytoskeleton and for regulating cell migration by integrins and non-muscle myosin II.

alpha4 beta1-Integrin regulates directionally persistent cell migration in response to shear flow stimulation.

alpha(4)beta(1)-Integrin plays a pivotal role in cell migration in vivo. This integrin has been shown to regulate the front-back polarity of migrating cells via localized inhibition of alpha(4)-integrin/paxillin binding by phosphorylation at the alpha(4)-integrin cytoplasmic tail. Here, we demonstrate that alpha(4)beta(1)-integrin regulates directionally persistent cell migration via a more complex mechanism in which alpha(4)-integrin phosphorylation and paxillin binding act via both cooperative and independent pathways. We show that, in response to shear flow, alpha(4)beta(1)-integrin binding to the CS-1 region of fibronectin was necessary and sufficient to promote directionally persistent cell migration when this integrin was ectopically expressed in CHO cells. Under shear flow, the alpha(4)beta(1)-integrin-expressing cells formed a fan shape with broad lamellipodia at the front and retracted trailing edges at the back. This "fanning" activity was enhanced by disrupting paxillin binding alone and inhibited by disrupting phosphorylation alone or together with disrupting paxillin binding. Notably, the phosphorylation-disrupting mutation and the double mutation resulted in the formation of long trailing tails, suggesting that alpha(4)-integrin phosphorylation is required for trailing edge retraction/detachment independent of paxillin binding. Furthermore, the stable polarity and directional persistence of shear flow-stimulated cells were perturbed by the double mutation but not the single mutations alone, indicating that paxillin binding and alpha(4)-integrin phosphorylation can facilitate directionally persistent cell migration in an independent and compensatory manner. These findings provide a new insight into the mechanism by which integrins regulate directionally persistent cell migration.

Golgin-160 promotes cell surface expression of the beta-1 adrenergic receptor.

Golgin-160 is a ubiquitously expressed peripheral Golgi membrane protein that is important for transduction of certain pro-apoptotic signals at the Golgi complex. However, the role of golgin-160 in normal Golgi structure and function is unknown. Here, we show that depletion of golgin-160 using RNA interference (RNAi) does not affect Golgi morphology or constitutive membrane traffic in HeLa cells. However, depletion of golgin-160 leads to significantly decreased cell surface levels of exogenously expressed beta1-adrenergic receptor (beta1AR), which can be rescued by expression of RNAi-resistant forms of golgin-160. Furthermore, overexpression of golgin-160 leads to higher surface levels of beta1AR. Golgin-160 is localized mostly in the cis and medial regions of the Golgi stack by immunoelectron microscopy, suggesting that it does not directly promote incorporation of beta1AR into transport vesicles at the trans Golgi network. Golgin-160 interacts with beta1AR in vitro, and we mapped the interaction to a region between residues 140 and 257 in the head of golgin-160 and the third intracellular loop of beta1AR. Our results support the idea that golgin-160 may promote efficient surface delivery of a subset of cargo molecules.

Comparative proteome analysis of laboratory grown Brucella abortus 2308 and Brucella melitensis 16M.

Brucella species are pathogenic agents that cause brucellosis, a debilitating zoonotic disease that affects a large variety of domesticated animals and humans. Brucella melitensis and Brucella abortus are considered major health threats because of their highly infectious nature and worldwide occurrence. The availability of the annotated genomes for these two species has allowed a comparative proteomics study of laboratory grown B. melitensis 16M and B. abortus 2308 by two-dimensional (2-D) gel electrophoresis and peptide mass fingerprinting. Computer-assisted analysis of the different 2-D gel images of strains 16M and 2308 revealed significant quantitative and qualitative differences in their protein expression patterns. Proteins involved in membrane transport, particularly the high affinity amino acids binding proteins, and those involved in Sec-dependent secretion systems related to type IV and type V secretion systems, were differentially expressed. Differential expression of these proteins may be responsible for conferring specific host preference in the two strains 2308 and 16M.

Brucella proteomes--a review.

The proteomes of selected Brucella spp. have been extensively analyzed by utilizing current proteomic technology involving 2-DE and MALDI-MS. In Brucella melitensis, more than 500 proteins were identified. The rapid and large-scale identification of proteins in this organism was accomplished by using the annotated B. melitensis genome which is now available in the GenBank. Coupled with new and powerful tools for data analysis, differentially expressed proteins were identified and categorized into several classes. A global overview of protein expression patterns emerged, thereby facilitating the simultaneous analysis of different metabolic pathways in B. melitensis. Such a global characterization would not have been possible by using time consuming and traditional biochemical approaches. The era of post-genomic technology offers new and exciting opportunities to understand the complete biology of different Brucella species.

Comparative proteome analysis of Brucella melitensis vaccine strain Rev 1 and a virulent strain, 16M.

The genus Brucella consists of bacterial pathogens that cause brucellosis, a major zoonotic disease characterized by undulant fever and neurological disorders in humans. Among the different Brucella species, Brucella melitensis is considered the most virulent. Despite successful use in animals, the vaccine strains remain infectious for humans. To understand the mechanism of virulence in B. melitensis, the proteome of vaccine strain Rev 1 was analyzed by two-dimensional gel electrophoresis and compared to that of virulent strain 16M. The two strains were grown under identical laboratory conditions. Computer-assisted analysis of the two B. melitensis proteomes revealed proteins expressed in either 16M or Rev 1, as well as up- or down-regulation of proteins specific for each of these strains. These proteins were identified by peptide mass fingerprinting. It was found that certain metabolic pathways may be deregulated in Rev 1. Expression of an immunogenic 31-kDa outer membrane protein, proteins utilized for iron acquisition, and those that play a role in sugar binding, lipid degradation, and amino acid binding was altered in Rev 1.

Global analysis of the Brucella melitensis proteome: Identification of proteins expressed in laboratory-grown culture.

Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.