Rabphilin localizes with the cell actin cytoskeleton and stimulates association of granules with F-actin cross-linked by {alpha}-actinin.
In endocrine cell, granules accumulate within an F-actin-rich region below the plasma membrane. The mechanisms involved in this process are largely unknown. Rabphilin is a cytosolic protein that is expressed in neurons and neuroendocrine cells and binds with high affinity to members of the Rab3 family of GTPases localized to synaptic vesicles and dense core granules. Rabphilin also interacts with alpha-actinin, a protein that cross-links F-actin into bundles and networks and associates with the granule membrane. Here we asked whether rabphilin, in addition to its granule localization, also interacts with the cell actin cytoskeleton. Immunofluorescence and immunoelectron microscopy show that rabphilin localizes to the sub-plasmalemmal actin cytoskeleton both in neuroendocrine and unspecialized cells. By using purified components, it is found that association of rabphilin with F-actin is dependent on added alpha-actinin. In an in vitro assay, granules, unlike endosomes or mitochondria, associate with F-actin cross-linked by alpha-actinin. Rabphilin is shown to stimulate this process. Rabphilin enhances by approximately 8-fold the granule ability to localize within regions of elevated concentration of cross-linked F-actin. These results suggest that rabphilin, by interacting with alpha-actinin, organizes the cell cytoskeleton to facilitate granule localization within F-actin-rich regions.
Baldini
Giovanna
G
Dipartimento di Morfologia Umana Normale, via Manzoni 16, Trieste, Universita' di Trieste, Trieste I-34138, Italy. gbaldini@uams.edu
Martelli
Alberto M
AM
Tabellini
Giovanna
G
Horn
Chad
C
Machaca
Khaled
K
Narducci
Paola
P
Baldini
Giulia
G
D000185:Actinin / Q000737:chemistry* / Q000378:metabolism; D000199:Actins / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002522:Chlorocebus aethiops; D003599:Cytoskeleton / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015151:Immunoblotting; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008928:Mitochondria / Q000378:metabolism; D009474:Neurons / Q000378:metabolism; D016716:PC12 Cells; D011485:Protein Binding; D051381:Rats; D014162:Transfection; D020692:rab3 GTP-Binding Proteins / Q000378:metabolism / Q000502:physiology*
D000199:Actins; D000185:Actinin; D049452:Green Fluorescent Proteins; D005982:Glutathione Transferase; C492132:Rab3b protein, rat; D020692:rab3 GTP-Binding Proteins