AmpA protein functions by different mechanisms to influence early cell type specification and to modulate cell adhesion and actin polymerization in Dictyostelium discoideum

At the aggregation stage of Dictyostelium discoideum development, a cell surface glycoprotein of Mr 80,000 (gp80) has been found to mediate the EDTA-resistant type of cell-cell adhesion via homophilic interaction (Siu, C.-H., A. Cho, and A. H. C. Choi. 1987. J. Cell Biol. 105:2523-2533). To investigate the structure-function relationships of gp80, we have isolated full length cDNA clones for gp80 and determined the DNA sequence. The deduced structure of gp80 showed three major domains. An amino-terminal globular domain composed of the bulk of the protein is supported by a short stalk region, which is followed by a membrane anchor at the carboxy terminus. Structural analysis suggested that the cell-binding domain of gp80 resides within the globular domain near the amino terminus. To investigate the relationship of the cell-binding activity to this region of the polypeptide, three protein A/gp80 (PA80) gene fusions were constructed using the expression vector pRIT2T. These PA80 fusion proteins were assayed for their ability to bind to aggregation stage cells. Binding of 125I-labeled fusion proteins PA80I (containing the Val123 to Ile514 fragment of gp80) and PA80II (Val123 to Ala258) was dosage dependent and could be inhibited by precoating cells with the cell cohesion-blocking mAb 80L5C4. On the other hand, there was no appreciable binding of PA80III (Ile174 to Ile514) to cells. Reassociation of cells was significantly inhibited in the presence of PA80I or PA80II. In addition, 125I-labeled PA80II exhibited homophilic interaction with immobilized PA80I, PA80II, or gp80. The results of these studies lead to the mapping of a cell-binding domain in the region between Val123 and Leu173 of gp80 and provide direct evidence that the cell-binding activity of gp80 resides in the protein moiety.

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Structural Basis for Partial Redundancy in a Class of Transcription Factors, the LIM Homeodomain Proteins, in Neural Cell Type Specification

Journal of Biological Chemistry ◽

10.1074/jbc.m111.248559 ◽

2011 ◽

Vol 286 (50) ◽

pp. 42971-42980 ◽

Cited By ~ 28

Author(s):

Morgan S. Gadd ◽

Mugdha Bhati ◽

Cy M. Jeffries ◽

David B. Langley ◽

Jill Trewhella ◽

...

Keyword(s):

Transcription Factors ◽

Neural Cell ◽

Structural Basis ◽

Cell Type ◽

Homeodomain Proteins ◽

Partial Redundancy ◽

Type Specification ◽

Lim Homeodomain

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Erratum to: The L1 Family of Cell Adhesion Molecules: A Sickening Number of Mutations and Protein Functions

Advances in Neurobiology - Cell Adhesion Molecules ◽

10.1007/978-1-4614-8090-7_15 ◽

2013 ◽

pp. E1-E1

Author(s):

Kakanahalli Nagaraj ◽

Rula Mualla ◽

Michael Hortsch

Keyword(s):

Cell Adhesion ◽

Adhesion Molecules ◽

Cell Adhesion Molecules ◽

Protein Functions

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Evolutionary Perspectives on the Moonlighting Functions of Bacterial Factors That Support Actin-Based Motility

mBio ◽

10.1128/mbio.01520-19 ◽

2019 ◽

Vol 10 (4) ◽

Cited By ~ 1

Author(s):

Volkan K. Köseoğlu ◽

Hervé Agaisse

Keyword(s):

Cell Adhesion ◽

Host Cell ◽

Biofilm Formation ◽

Actin Polymerization ◽

Current Knowledge ◽

Bacterial Pathogens ◽

Actin Nucleation ◽

Bacterial Aggregation ◽

Moonlighting Functions ◽

Evolutionary Perspectives

ABSTRACT Various bacterial pathogens display an intracellular lifestyle and spread from cell to cell through actin-based motility (ABM). ABM requires actin polymerization at the bacterial pole and is mediated by the expression of bacterial factors that hijack the host cell actin nucleation machinery or exhibit intrinsic actin nucleation properties. It is increasingly recognized that bacterial ABM factors, in addition to having a crucial task during the intracellular phase of infection, display “moonlighting” adhesin functions, such as bacterial aggregation, biofilm formation, and host cell adhesion/invasion. Here, we review our current knowledge of ABM factors and their additional functions, and we propose that intracellular ABM functions have evolved from ancestral, extracellular adhesin functions.

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Simultaneous localization and quantification of relative G and F actin content: optimization of fluorescence labeling methods.

Journal of Histochemistry & Cytochemistry ◽

10.1177/40.10.1527379 ◽

1992 ◽

Vol 40 (10) ◽

pp. 1605-1612 ◽

Cited By ~ 68

Author(s):

G C Knowles ◽

C A McCulloch

Keyword(s):

Flow Cytometry ◽

Confocal Microscopy ◽

Actin Polymerization ◽

Direct Method ◽

Dnase I ◽

Fluorescence Probes ◽

Fluorescence Labeling ◽

Cell Type ◽

Fluorescence Spectrophotometry ◽

Actin Rearrangement

Previous studies of fluorescence probes for labeling the monomeric actin pool have demonstrated lack of specificity. We have used quantitative analytical methods to assess the sensitivity and specificity of rhodamine DNAse I as a probe for monomeric (G) actin. The G-actin pool of attached or suspended fibroblasts was stabilized by ice-cold glycerol and MgCl2. Formaldehyde fixation was used to clamp the filamentous (F) actin pool. G- and F-actins were stained by rhodamine DNAse I and FITC-phalloidin, respectively. Confocal microscopy indicated that the G- and F-actins were spatially separate in substrate-attached cells. Flow cytometry and fluorescence spectrophotometry demonstrated low co-labeling of the separate actin pools, although measureable background binding of rhodamine DNAse I was detectable. Estimates of the extent of actin polymerization after trypsinization demonstrated reciprocal changes of monomeric and filamentous actins, consistent with the formation of a perinuclear array of F-actin. The labeling and quantitation methods were also sufficiently sensitive to detect cell type-dependent variations in actin content. Dual labeling of cells with rhodamine DNAse I and FITC-phalloidin may provide a simple and direct method to image and quantify actin rearrangement in individual cells.

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