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First published online 1 April 2003
doi: 10.1242/jcs.00398

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Cytoplasmic dynein participates in apically targeted stimulated secretory traffic in primary rabbit lacrimal acinar epithelial cells

¹ Department of Pharmaceutical Sciences, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, USA
² Department of Physiology and Biophysics, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, USA
³ Department of Ophthalmology, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, USA
⁴ Department of Pathology, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, USA
⁵ Institute for Genetic Medicine, University of Southern California, 1985 Zonal Avenue, Los Angeles, CA 90033, USA

View larger version (138K): [in a new window]   Fig. 1. CCH promotes MT-dependent recruitment of dynein to the subapical region of acini. Lacrimal acini were fixed and processed for labeling of dynein and actin filaments, and imaged by confocal fluorescence microscopy. Dynein (green) is shown in panels A, D, G and J, actin filaments (red) in the same samples in B, E, H and K, and overlaid images in C, F, I and L. (A-C) resting acini; (D-F) CCH-stimulated acini (100 µM, 15 minutes); (G-I) nocodazole-treated acini (33 µM, 60 minutes); (J-L) nocodazole-treated acini (33 µM, 60 minutes) exposed to CCH (100 µM, 15 minutes). Asterisk, lumenal regions; arrowhead, punctate labeling. Bar, 10 µm.

View larger version (183K): [in a new window]   Fig. 2. CCH promotes recruitment of p150Glued and Arp1 to the subapical region of acini. Lacrimal acini were fixed and processed for labeling of p150Glued or Arp1 with actin filaments and imaged by confocal fluorescence microscopy. Panels A and B show p150Glued (green) and actin filaments (red) in resting and CCH-stimulated (100 µM, 15 minutes) lacrimal acini, respectively, whereas panels C and D show Arp1 (green) and actin filaments (red) in resting and CCH-stimulated (100 µM, 15 minutes) lacrimal acini, respectively. Asterisk, lumenal regions. Bar, 10 µm.

View larger version (28K): [in a new window]   Fig. 3. CCH increases recovery of p150Glued and Arp1 in Triton X-100 insoluble, SDS soluble fractions. Lacrimal acini were treated without (Con) or with CCH (100 µM, 15 minutes) before sequential extraction into buffers containing saponin (Sap), Tx-100 and SDS. (A) Western blots of detergent eluates from representative preparations of Con and CCH-treated lacrimal acini showing p150Glued, Arp1 and DIC contents in each pool. Blots used appropriate primary and IRDyeTM800-conjugated secondary antibodies. Equal volumes of each pool were loaded, with protein assays confirming that CON and CCH detergent eluates within each pool had equivalent protein contents. (B-D) Summary of changes in p150Glued, Arp1 and DIC recovery in each eluate from Con and CCH-stimulated acini expressed as a percentage of the total cellular content of each protein. n=7-10 preparations; error bars represent s.e.m.; *Significant increase at P<0.05; #significant decrease at P<0.05.

View larger version (97K): [in a new window]   Fig. 4. CCH increases dynein and dynactin colocalization with VAMP2 in the subapical region of acini. Lacrimal acini were fixed and processed to label DIC, p150Glued or Arp1 with rab3D or VAMP2 and imaged by confocal fluorescence microscopy. (A-L) Resting acini, whereas A'-L' depict CCH-stimulated (100 µM, 15 minutes) acini. A-C and A'-C' show DIC (green) and VAMP2 (red) separately (A,A',B,B') and overlaid (C,C'); D-F and D'-F' show Arp1 (green) and VAMP2 (red) separately (D,D',E,E') and overlaid (F,F'); G-I and G'-I' show p150Glued (green) and VAMP2 (red) separately (G,G',H,H') and overlaid (I,I'); and J-L and J'-L' show p150Glued (green) and rab3D (red) separately (J,J',K,K') and overlaid (L,L'). Arrows and arrowheads, regions of colocalization of dynein or dynactin with VAMP2 or rab3D, respectively. Asterisk, lumenal regions. Bar, 10 µm.

View larger version (28K): [in a new window]   Fig. 5. MT-affinity isolation of subcellular membranes from resting and CCH-stimulated acini. Lacrimal acini were incubated without (Con) and with CCH (100 µM, 15 minutes) and processed to isolate membranes using MT-affinity and release with excess ATP. Western blots using appropriate primary and IRDyeTM800-conjugated secondary antibodies from a representative preparation are shown in A, whereas B depicts summary data showing the contents of DIC and VAMP2 in MT-affinity, ATP release fractions under each condition. 40 µg of protein from resting and CCH-stimulated membrane samples were loaded under each condition. n=4-5 separate preparations. *Significant increase at P0.05.

View larger version (25K): [in a new window]   Fig. 6A. Dynein, dynactin and secretory and biosynthetic compartment markers after density gradient analysis of resting and CCH-stimulated acini. Lacrimal acini incubated without (resting) and with CCH (100 µM, 15 minutes) were processed by isopycnic centrifugation on sorbitol density gradients. The distributions of p150Glued, Arp1, DIC, ß-hexosaminidase and total protein.

View larger version (24K): [in a new window]   Fig. 6B. Dynein, dynactin and secretory and biosynthetic compartment markers after density gradient analysis of resting and CCH-stimulated acini. Lacrimal acini incubated without (resting) and with CCH (100 µM, 15 minutes) were processed by isopycnic centrifugation on sorbitol density gradients. The distributions of VAMP2, -adaptin, acid phosphatase, rab3D and rab6. Fractions were pooled for VAMP2 and -adaptin to conserve material for western blotting. Left and middle columns show distributions of markers from resting and CCH-stimulated acini, respectively, whereas the right columns show CCH-induced changes. n=3-8 preparations; error bars represent s.e.m. *P<0.05.

View larger version (82K): [in a new window]   Fig. 7. Dynein, dynactin and membrane compartment markers after phase partitioning analysis of density gradient fractions. Density gradient fractions from CCH-stimulated acini were analyzed by partitioning in an aqueous dextran-polyethyleneglycol two-phase system. Gradient fractions 1 and P were analyzed individually; other fractions were pooled as follows: 4+5, 7+8, 9+10, 11+12. Modal positions of compartments are designated by a-o. Compartments j and m are proposed to contain secretory transport vesicles and related compartments [recruitable secretory vesicles (rsv) and immature secretory vesicles (isv)]. Other compartment designations are discussed in detail by Qian et al. (Qian et al., 2002). (a) Basal-lateral recycling endosome; (e) Basal-lateral sorting endosome; (ß-d,f,h,i) Specialized TGN microdomains; (g) Golgi complex; (k) Secretory vesicle membrane fragments; (l,o) Pre-lysosomes; (n) Lysosomes. Similar results were obtained in a second analysis with a different phase system composition.

View larger version (59K): [in a new window]   Fig. 8. Transduction of acini with Ad constructs. (A,B) Phase microscopy images of untransduced and Ad-LacZ-transduced acini, respectively, processed for detection of ß-galactosidase activity with X-Gal as substrate. (C,D) Fluorescence microscopy images of untransduced and Ad-GFP-transduced acini, respectively. (E,F) Confocal fluorescence microscopy images of untransduced and Ad-Dynt-transduced acini, respectively, processed for detection of dynamitin by immunofluorescence. (G) The spectrum of 35S-labeled proteins obtained by autoradiography of proteins from control and transduced acini resolved by SDS-PAGE. Arrows indicate positions of ß-galactosidase (ß-Gal), GFP and dynamitin (p50). H shows dynamitin content in lysates from untransduced (Con), Ad-Dynt-transduced and Ad-Dynt-GFP transduced acini by western blotting. Transduction with constructs was at a MOI of 5 PFU/cell for 4 hours followed by rinsing and recovery for 16-18 hours. Bars in D and F represent 10 µm and reflect magnifications in A-D and E-F, respectively. Asterisk, lumenal regions.

View larger version (156K): [in a new window]   Fig. 9. Dynamitin overexpression inhibits CCH-stimulated recruitment of DIC, p150Glued and VAMP2 to the subapical region. Resting and CCH-stimulated (100 µM, 15 minutes) lacrimal acini transduced with Ad-LacZ or Ad-Dynt were fixed and processed for detection of p150Glued (green, A-A',C-C',E-E',G-G') or DIC (green, B-B',D-D',F-F',H-H') with actin filaments (red, A-D,A'-D') or VAMP2 (red, E-H,E'-H') and imaged by confocal fluorescence microscopy. (A-H,A'-H') Resting and CCH-stimulated (100 µM, 15 minutes) acini, respectively. A-A', B-B', E-E' and F-F' were transduced with Ad-LacZ and C-C', D-D', G-G' and H-H' were transduced with Ad-Dynt. Arrowheads, punctate labeling; arrows, colocalization of DIC or p150Glued with VAMP2; asterisk, lumenal regions. Bar, 10 µm.

View larger version (111K): [in a new window]   Fig. 10. Dynamitin overexpression promotes increased cytoplasmic colocalization of VAMP2 and Arp1 in CCH-stimulated acini. Resting and CCH-stimulated lacrimal acini transduced with Ad-LacZ or Ad-Dynt were fixed and processed for detection of Arp1 (A-A' and B-B') and VAMP2 (C-C' and D-D'). (E-E' and F'F') Colocalization of these markers. (A-A', C-C' and E'E') Acini transduced with Ad-LacZ; (B-B', D-D' and F-F') Acini transduced with Ad-Dynt. (A-F) Resting acini; (A'-F') CCH-stimulated (100 µM, 15 minutes) acini. Bar, 10 µm. (*) Lumenal regions; arrowheads, colocalization of Arp1 and VAMP2 in the cytoplasm; arrows, colocalization of Arp1 and VAMP2 beneath the apical membrane.

View larger version (139K): [in a new window]   Fig. 11. Dynamitin overexpression or nocodazole treatment reduce the subapical accumulation of rab3D in resting acini. Untransduced (Con) and transduced rabbit lacrimal acini were fixed and processed for detection of rab3D (green, A-C,E,F) or MTs (green, G and H) in parallel with actin filaments (red). (A,B,C) rab3D and actin filaments in untransduced, Ad-LacZ-transduced and Ad-Dynt-transduced acini, respectively. (D) rab3D distribution quantified into three categories from 366-404 lumena from 3-4 separate preparations under each condition: apical, half apical and fully dispersed. (E,F) rab3D labeling beneath actin-enriched lumenal regions in untreated and nocodazole-treated (33 µM, 60 minutes) acini, respectively. Arrowheads indicate rab3D-enriched secretory vesicles. (G,H) MTs and actin filaments in Ad-LacZ-transduced and Ad-Dynt-transduced acini, respectively. Arrows indicate MTs extending from the apical pole; asterisk, lumenal regions. Bar in F, 6 µm. Bar in H, 10 µm.

View larger version (27K): [in a new window]   Fig. 12. Dynamitin overexpression modifies CCH-independent and CCH-dependent release of bulk protein and ß-hexosaminidase. Lacrimal acini cultured on Matrigel-coated 24-well plates were transduced with Ad-GFP or Ad-Dynt-GFP, treated without or with CCH (100 µM, 30 minutes) and processed for measurement of basal (Bas), total (Tot) and stimulated (Stim) protein and ß-hexosaminidase release as described. n=6 preparations; *P0.05; A.U., arbitrary fluorescence unit.