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Ndm, a coiled-coil domain protein that suppresses macropinocytosis and has effects on cell migration.

ABSTRACT: The ampA gene has a role in cell migration in Dictyostelium discoideum. Cells overexpressing AmpA show an increase in cell migration, forming large plaques on bacterial lawns. A second-site suppressor of this ampA-overexpressing phenotype identified a previously uncharacterized gene, ndm, which is described here. The Ndm protein is predicted to contain a coiled-coil BAR-like domain-a domain involved in endocytosis and membrane bending. ndm-knockout and Ndm-monomeric red fluorescent protein-expressing cell lines were used to establish a role for ndm in suppressing endocytosis. An increase in the rate of endocytosis and in the number of endosomes was detected in ndm(-) cells. During migration ndm(-) cells formed numerous endocytic cups instead of the broad lamellipodia structure characteristic of moving cells. A second lamellipodia-based function-cell spreading-was also defective in the ndm(-) cells. The increase in endocytosis and the defect in lamellipodia formation were associated with reduced chemotaxis in ndm(-) cells. Immunofluorescence results and glutathione S-transferase pull-down assays revealed an association of Ndm with coronin and F-actin. The results establish ndm as a gene important in regulating the balance between formation of endocytic cups and lamellipodia structures.

SUBMITTER: Kelsey JS

PROVIDER: S-EPMC3431939 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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Ndm, a coiled-coil domain protein that suppresses macropinocytosis and has effects on cell migration.

Kelsey Jessica S JS Fastman Nathan M NM Noratel Elizabeth F EF Blumberg Daphne D DD

Molecular biology of the cell 20120718 17

The ampA gene has a role in cell migration in Dictyostelium discoideum. Cells overexpressing AmpA show an increase in cell migration, forming large plaques on bacterial lawns. A second-site suppressor of this ampA-overexpressing phenotype identified a previously uncharacterized gene, ndm, which is described here. The Ndm protein is predicted to contain a coiled-coil BAR-like domain-a domain involved in endocytosis and membrane bending. ndm-knockout and Ndm-monomeric red fluorescent protein-expre ...[more]

PMID: 22809629

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Project description:Two types of viruses are produced during the baculovirus life cycle: budded virus (BV) and occlusion-derived virus (ODV). A particular baculovirus protein, FP25K, is involved in the switch from BV to ODV production. Previously, FP25K from the model alphabaculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) was shown to traffic ODV envelope proteins. However, FP25K localization and the domains involved are inconclusive. Here we used a quantitative approach to study FP25K subcellular localization during infection using an AcMNPV bacmid virus that produces a functional AcMNPV FP25K-green fluorescent protein (GFP) fusion protein. During cell infection, FP25K-GFP localized primarily to the cytoplasm, particularly amorphous structures, with a small fraction being localized in the nucleus. To investigate the sequences involved in FP25K localization, an alignment of baculovirus FP25K sequences revealed that the N-terminal putative coiled-coil domain is present in all alphabaculoviruses but absent in betabaculoviruses. Structural prediction indicated a strong relatedness of AcMNPV FP25K to long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p), which contains an N-terminal coiled-coil domain responsible for cytoplasmic retention. Point mutations and deletions of this domain lead to a change in AcMNPV FP25K localization from cytoplasmic to nuclear. The coiled-coil and C-terminal deletion viruses increased BV production. Furthermore, a betabaculovirus FP25K protein lacking this N-terminal coiled-coil domain localized predominantly to the nucleus and exhibited increased BV production. These data suggest that the acquisition of this N-terminal coiled-coil domain in FP25K is important for the evolution of alphabaculoviruses. Moreover, with the divergence of preocclusion nuclear membrane breakdown in betabaculoviruses and membrane integrity in alphabaculoviruses, this domain represents an alphabaculovirus adaptation for nuclear trafficking of occlusion-associated proteins.ImportanceBaculovirus infection produces two forms of viruses: BV and ODV. Manufacturing of ODV involves trafficking of envelope proteins to the inner nuclear membrane, mediated partly through the FP25K protein. Since FP25K is present in alpha-, beta-, and gammabaculoviruses, it is uncertain if this trafficking function is conserved. In this study, we looked at alpha- and betabaculovirus FP25K trafficking by its localization. Alphabaculovirus FP25K localized primarily to the cytoplasm, whereas betabaculovirus FP25K localized to the nucleus. We found that an N-terminal coiled-coil domain present in all alphabaculovirus FP25K proteins, but absent in betabaculovirus FP25K, was critical for alphabaculovirus FP25K cytoplasmic localization. We believe that this represents an evolutionary process that partly led to the gain of function of this N-terminal coiled-coil domain in alphabaculovirus FP25K to aid in nuclear trafficking of occlusion-associated proteins. Due to betabaculovirus breakdown of the nuclear membrane before occlusion, this function is not needed, and the domain was lost or never acquired.

Dataset Information

Ndm, a coiled-coil domain protein that suppresses macropinocytosis and has effects on cell migration.

Publications

Ndm, a coiled-coil domain protein that suppresses macropinocytosis and has effects on cell migration.

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