These findings highlight the importance of further studies to detail the exact function of MTs in morphogenesis. Supporting Information Figure S1 (A) Polyclonal MDCK cells generated by lentiviral gene transfer Tilbroquinol to express tetracycline inducible shRNA against Kif3a and a GFP reporter are subjected to western blot analysis 24 h and 48 h hours after seeding. Western Blot of lysates from MDCK.Kif3a-i cells upon tetracycline treatment demonstrate downregulation of Kap, but same levels of acetylated-and detyrosinated tubulin. (E) Migrating MDCK cells were stained for APC (green), -tubulin (magenta), and nuclei (blue). Punctuate staining of APC is present at plus-ends of MTs in Kif3a-i cells without tetracycline treatment (-Tet), but not in Kif3a-i depleted cells (+Tet). Scale Bars: 10 m.(TIF) pone.0062165.s001.tif (2.4M) GUID:?20BF3259-40AB-4D86-B640-8931D9EEE28B Figure S2: (A) Staining with antibodies against Zo-1, E-Cadherin, -Catenin, Par3 and Scrib (white) reveals similar patterns in Kif3a-i cells grown on solid supports, both in the absence (-Tet) and presence (+Tet) of tetracycline. GFP appears in the merged image when Kif3a-i cells express the shRNA construct. Central Par3 signal represents staining Tilbroquinol at the mother centriole, as it has been described In the Par3 and Scrib stained panels GFP fluorescence is absent due to fixation with methanol. Scale bars: 20 m. Lower images: Kif3a-i cells were stably transduced with the apical protein Kim1-CFP. Confocal z-stacks and xz-sectioning reveal localization of Kim1-CFP at the apical membrane. Scale Bars: 10 m.(TIF) pone.0062165.s002.tif (7.0M) GUID:?9858E15E-A7E8-45BA-8643-27FE775F9849 Table S1: Quantification of microtubule behaviour in MDCK.Kif3a-i cells without or with tetracycline. (XLSX) pone.0062165.s003.xlsx (11K) GUID:?759E1D9B-4A40-446C-B6F1-8784B7893EFF Video S1: Sheet Migration in Kif3a-deficient cells ( Figure 1 ). Kif3a-i cells grown to confluence for two days without tetracycline (-Tet, left) close the wound after injury of the monolayer. Kif3a deficient cells (+Tet, right) have a severe migration defect (phase contrast images).(MOV) pone.0062165.s004.mov (909K) GUID:?F78F38CF-9BDD-4EC7-A10E-AC1B11C1C059 Video S2: Kif3a-YFP associates with microtubule plus-ends during migration in MDCK cells. MDCK cells were stably transduced with Kif3a-YFP and -Tubulin-CFP, grown to confluence for two days, injured and migrating cells (six hours after wounding) imaged by dual camera TIRF microscopy. Kif3a-YFP signals are seen along MTs and at plus ends of MTs. Images were collected every two seconds for three minutes.(MOV) pone.0062165.s005.mov (666K) GUID:?FC0B53EA-6BF9-4AFC-95F0-B0504AACE52F Video S3: Eb1-YFP in migrating MDCK.Kif3a-i cells. MDCK.Kif3a-i cells expressing Eb1-YFP cells were incubated with or without Tet and Eb1 dynamics were assessed in migrating cells six hours after wounding. In Tilbroquinol the absence of Tet Eb1-tracks lead perpendicularly into the leading edge, but they move parallel to the wound margin in Kif3a depleted cells.(MOV) pone.0062165.s006.mov (1.2M) GUID:?72EB0975-1F82-4D70-BF94-EF9633A13609 Video S4: Microtubule dynamics in migrating MDCK.Kif3a-i cells. MDCK.Kif3a-i cells expressing -Tubulin-YFP cells were incubated with or without Tet and microtubules were observed in migrating cells six hours after wounding to quantify MT dynamics. The MT ends appear stunned in Kif3a-deficient cells.(MOV) pone.0062165.s007.mov (1.9M) GUID:?77278D68-2843-418A-BE80-73F282814B1D Abstract The microtubular motor Kinesin-2 and its subunit Kif3a are essential for the formation of primary cilia, an organelle implicated in a wide spectrum of developmental abnormalities. Outside cilia, Tilbroquinol Kinesin-2 mediated transport has been implicated in vesicle and N-cadherin transport, but it is unknown if and how extraciliary Kif3a affects basic cellular functions such as migration or the formation of multicellular structures. Here we show that tetracycline inducible depletion of Kif3a in MDCK cells slows epithelial cell migration. Microtubules at the leading edge of Kif3a depleted cells failed to grow perpendicularly into the leading edge and microtubular dynamics were dampened in Kif3a depleted cells. Loss of Kif3a retarded lateral membrane specification and completely prevented the formation of three-dimensional spheres in collagen. These data uncover that Kif3a regulates the microtubular cytoskeleton in the cell periphery and imply that extra-ciliary Kif3a has an unexpected function in morphogenesis. Introduction Kif3a is a subunit of hetero-trimeric Kinesin-2, a microtubule (MT) plus-end directed motor protein . Hetero-trimeric Kinesin-2 consists of the two motor subunits Kif3a/Kif3b and the cargo-binding protein Kap3 . Kinesin-2 has been mostly studied in cilia where it functions to carry protein complexes along the microtubular axoneme from the base Rabbit Polyclonal to MMP-11 to the tip of the cilium in a process termed intraflagellar transport (IFT) that is required for cilia formation . The constitutive deletion of Kif3a in mice results in severely disordered mesoderm development reminiscent of defective hedgehog signalling and in mid-embryonic lethality , . Several Hedgehog (HH) components localize to cilia and this organelle plays a central role in the activation of the HH pathway which could explain.