Protein kinase D-mediated anterograde membrane trafficking is required for fibroblast motility.
BACKGROUND
Locomoting cells exhibit a constant retrograde flow of plasma membrane (PM) proteins from the leading edge lamellipodium backward, which when coupled to substrate adhesion, may drive forward cell movement. However, the intracellular source of these PM components and whether their continuous retrograde flow is required for cell motility is unknown.


RESULTS
To test the hypothesis that the anterograde secretion pathway supplies PM components for retrograde flow that are required for lamellipodial activity and cell motility, we specifically inhibited transport of cargo from the trans-Golgi network (TGN) to the PM in Swiss 3T3 fibroblasts and monitored cell motility using time-lapse microscopy. TGN-to-PM trafficking was inhibited with a dominant-negative, kinase-dead (kd) mutant of protein kinase D1 (PKD) that specifically blocks budding of secretory vesicles from the TGN and does not affect other transport pathways. Inhibition of PKD on the TGN inhibited directed cell motility and retrograde flow of surface markers and filamentous actin, while inhibition of PKD elsewhere in the cell neither blocked anterograde membrane transport nor cell motile functions. Exogenous activation of Rac1 in PKD-kd-expressing cells restored lamellipodial dynamics independent of membrane traffic. However, lamellipodial activity was delocalized from a single leading edge, and directed cell motility was not fully recovered.


CONCLUSIONS
These results indicate that PKD-mediated anterograde membrane traffic from the TGN to the PM is required for fibroblast locomotion and localized Rac1-dependent leading edge activity. We suggest that polarized secretion transmits cargo that directs localized signaling for persistent leading edge activity necessary for directional migration.
Prigozhina
Natalie L
NL
Department Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Road, CB163, La Jolla, CA 92037, USA.
Waterman-Storer
Clare M
CM
D000818:Animals; D002465:Cell Movement / Q000502:physiology*; D005347:Fibroblasts / Q000502:physiology*; D049452:Green Fluorescent Proteins; D007150:Immunohistochemistry; D007202:Indicators and Reagents / Q000378:metabolism; D008164:Luminescent Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000502:physiology*; D051379:Mice; D008845:Microinjections; D011493:Protein Kinase C / Q000378:metabolism / Q000502:physiology*; D011554:Pseudopodia / Q000502:physiology; D015398:Signal Transduction; D041701:Swiss 3T3 Cells; D020830:rac1 GTP-Binding Protein / Q000502:physiology; D021601:trans-Golgi Network / Q000502:physiology
D007202:Indicators and Reagents; D008164:Luminescent Proteins; D008565:Membrane Proteins; D049452:Green Fluorescent Proteins; C089212:protein kinase D; D011493:Protein Kinase C; D020830:rac1 GTP-Binding Protein