Cell Migration: A Physically Integrated Molecular Process

Proteomics, the large-scale analysis of proteins, will contribute greatly to our understanding of gene function in the post-genomic era. Proteomics can be divided into three main areas: (1) protein micro-characterization for large-scale identification of proteins and their post-translational modifications; (2) 'differential display' proteomics for comparison of protein levels with potential application in a wide range of diseases; and (3) studies of protein-protein interactions using techniques such as mass spectrometry or the yeast two-hybrid system. Because it is often difficult to predict the function of a protein based on homology to other proteins or even their three-dimensional structure, determination of components of a protein complex or of a cellular structure is central in functional analysis. This aspect of proteomic studies is perhaps the area of greatest promise. After the revolution in molecular biology exemplified by the ease of cloning by DNA methods, proteomics will add to our understanding of the biochemistry of proteins, processes and pathways for years to come.

/pdf/proteomics-to-study-genes-and-genomes-p3gszdgohu.pdf

Proteomics to study genes and genomes

Actin-Based Cell Motility and Cell Locomotion

4. Automated Interpretation of CID Spectra 282 5. Accurate Mass Tags 282 C. Protein Identification in Complex Mixtures 282 D. Analysis of Protein Expression 284 III. Proteomes and Post-Translational Modifications 285 A. Proteomes 285 1. The Analytical Challenge 285 2. Analysis of Protein−Protein Complexes 286 B. Post-Translational Modifications 286 1. Background 286 2. Detection and Purification of Phosphoproteins 288

Mass Spectrometry in Proteomics

Consensus sequences as substrate specificity determinants for protein kinases and protein phosphatases.

MOVEMENT of a eukaryotic cell along a substrate occurs by extension of lamellipodia and pseudopodia at the anterior and retraction at the posterior of the cell. The molecular and structural mechanisms of these movements are uncertain. Dictyostelium discoideum contains two forms of myosin. Here we show by immuno-fluorescence microscopy that non-filamentous myosin I occurs at the leading edges of the lamellipodial projections of migrating Dictyostelium amoebae, which are devoid of myosin II, whereas filamentous myosin II is concentrated in the posterior of the cells. On the basis of these locations of the two forms of myosin and their known biochemical and biophysical properties, we suggest that actomyosin I may contribute to the forces that cause extension at the leading edge of a motile cell, while the contraction of actomyosin II at the rear squeezes the cell mass forward. Myosin I isozymes might have similar roles in metazoan cells, for example at the leading edges of neuronal growth cones, and in the extension

Myosin I is located at the leading edges of locomoting Dictyostelium amoebae.

We report a fast, sensitive, and robust procedure for the identification of precise phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis by a combination of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI/TOF) and on-line capillary liquid chromatography electrospray tandem ion trap mass spectrometry (LC/ESI/MS/MS). With this procedure, a single phosphorylation site was identified on as little as 20 ng (500 fmol) of the baculovirus-expressed catalytic domain of myosin I heavy-chain kinase separated by gel electrophoresis. The phosphoprotein is digested in the gel with trypsin, and the resulting peptides are extracted with >60% yield and analyzed by MALDI/TOF before and after digestion with a phosphatase to identify the phosphopeptides. The phosphopeptides are then separated and fragmented in an on-line LC/ESI ion trap mass spectrometer to identify the precise phosphorylation sites. This procedure eliminates any off-line HPLC separation and mi...

Identification of phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis.

Acanthamoeba myosins IA and IB were localized by immunofluorescence and immunoelectron microscopy in vegetative and phagocytosing cells and the total cell contents of myosins IA, IB, and IC were quantified by immunoprecipitation. The quantitative distributions of the three myosin I isoforms were then calculated from these data and the previously determined localization of myosin IC. Myosin IA occurs almost exclusively in the cytoplasm, where it accounts for approximately 50% of the total myosin I, in the cortex beneath phagocytic cups and in association with small cytoplasmic vesicles. Myosin IB is the predominant isoform associated with the plasma membrane, large vacuole membranes and phagocytic membranes and accounts for almost half of the total myosin I in the cytoplasm. Myosin IC accounts for a significant fraction of the total myosin I associated with the plasma membrane and large vacuole membranes and is the only myosin I isoform associated with the contractile vacuole membrane. These data suggest that myosin IA may function in cytoplasmic vesicle transport and myosin I-mediated cortical contraction, myosin IB in pseudopod extension and phagocytosis, and myosin IC in contractile vacuole function. In addition, endogenous and exogenously added myosins IA and IB appeared to be associated with the cytoplasmic surface of different subpopulations of purified plasma membranes implying that the different myosin I isoforms are targeted to specific membrane domains through a mechanism that involves more than the affinity of the myosins for anionic phospholipids.

/pdf/differential-localization-of-acanthamoeba-myosin-i-isoforms-3vkk90g1px.pdf

Hanna Brzeska

Papers

Myosin I is located at the leading edges of locomoting Dictyostelium amoebae.

Identification of phosphorylation sites in proteins separated by polyacrylamide gel electrophoresis.

Differential localization of Acanthamoeba myosin I isoforms.

Regulation of class i and class ii myosins by heavy chain phosphorylation

The localization and sequence of the phosphorylation sites of Acanthamoeba myosins I: An improved method for locating the phosphorylated amino acid *