QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 12 February 2003
doi: 10.1242/jcs.00317

This Article Summary Full Text Full Text (PDF) Movie Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Rudolf, R. Articles by Gerdes, H.-H. Search for Related Content PubMed PubMed Citation Articles by Rudolf, R. Articles by Gerdes, H.-H. Social Bookmarking                         What's this?

Myosin Va facilitates the distribution of secretory granules in the F-actin rich cortex of PC12 cells

Rüdiger Rudolf¹, Tanja Kögel¹, Sergei A. Kuznetsov^1,*, Thorsten Salm¹, Oliver Schlicker¹, Andrea Hellwig¹, John A. Hammer, III² and Hans-Hermann Gerdes^1,

¹ Department of Neurobiology, Interdisciplinary Center of Neuroscience, University of Heidelberg, Im Neuenheimer Feld 364, D-69120 Heidelberg, Germany
² Laboratory of Cell Biology, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD 20892, USA
^* Present address: Institute of Cell Biology and Biosystems Technology, University of Rostock, Albert-Einstein Str. 3, D-18051 Rostock, Germany

View larger version (33K): [in a new window]   Fig. 5. Expression of FLAG-MCLT reduces the velocity of SGs. (A,B) PC12 cells, either single-transfected with hCgB-GFP(S65T) (control) or double-transfected with hCgB-GFP(S65T) and FLAG (FLAG) or FLAG-MCLT (FLAG-MCLT), respectively, were incubated for 2 hours at 20°C and then at 37°C as indicated. Cells were imaged at 37°C and the movements of SGs were tracked automatically. For each time point and condition at least 20 cells from four independent experiments were analysed. (A) Mean velocities of all SGs per cell are plotted as a function of chase time. Prior to plotting, the system-inherent error of the automated tracking algorithm used was subtracted from all values (see Materials and Methods). Error bars, s.e.m. (B) Frequency distributions of all velocity steps (n) recorded from frame to frame for each condition over the observation time indicated in A. Open arrowhead, maximal number of steps measured for control and FLAG-transfected cells; filled arrowhead, maximal number of steps measured for FLAG-MCLT-transfected cells. (C) PC12 cells, co-transfected with pcDNA3/PTS1-GFP and FLAG or FLAG-MCLT, respectively, were incubated for 2 hours at 20°C and then for 90 minutes at 37°C. Cells were imaged at 37°C and the movements of peroxisomes were tracked automatically. The frequency distributions of all velocity steps (n) are shown for each condition (C).

View larger version (85K): [in a new window]   Fig. 1. Myosin Va is distributed in the cortex of PC12 cells and colocalises with rCgB. (A-G) Non-transfected PC12 cells were cultured for 2 days in the absence (A-D) or presence (E-G) of NGF. Then, the cells were fixed and immunostained against rCgB (B,D,F) and myosin Va with DIL1 (A) and DIL2 (C,E) antibody, respectively. Single corresponding confocal sections (AB, CD, EF) are shown. G shows a Nomarski image of the same cells as in E and F. Arrowheads in A-D, show cortical subdomains enriched in both rCgB and myosin Va; arrows in E-G indicate growth cones. Bars, 10 µm. (H-H") Cells were transfected with hCgB-GFP(S65T). After incubation of cells for 2 hours at 20°C and then for 180 minutes at 37°C, a PNS was prepared and SGs were sedimented on coverslips by differential centrifugation, fixed, and immunostained with DIL2 antibody against myosin Va. Subsequently, single confocal sections of GFP-fluorescent SGs (H) and the corresponding immunosignals (H") were recorded. Arrows indicate immuno-positive, and arrowheads immuno-negative, GFP-fluorescent SGs (compare H and H'). Bar, 1 µm. The colocalisation of fluorescent SGs with myosin Va was quantified (H"). Black bar, colocalisation of GFP-fluorescence and immuno-signals form corresponding frame pairs (correct); white bar, random colocalisation of GFP-and immuno-signals from non-corresponding frame pairs (random). Error bars, s.e.m. (n=20 images, each 60x60 µm) corresponding to >500 fluorescent SGs.

View larger version (55K): [in a new window]   Fig. 2. Myosin Va is associated with SGs at the ultrastructural level. SGs from non-transfected PC12 cells were prepared by differential centrifugation (see Materials and Methods). SGs were then processed for immunoelectron microscopy in the absence (A) or presence (B) of anti-myosin Va, or with blocked anti-myosin Va (not shown), and protein A 10 nm immuno-gold (A,B). Bar, 500 nm. Unlabelled SGs (A, filled arrowheads) and labelled SGs (B, open arrowheads) are indicated. A quantification of the myosin Va immunolabelling of SGs is shown in C.

View larger version (55K): [in a new window]   Fig. 3. Myosin Va cofractionates with SGs on sucrose gradients. A PNS of non-transfected PC12 cells was subjected to differential and then to equilibrium sucrose gradient (0.8-2 M sucrose) centrifugation. Equal aliquots of the final equilibrium gradient fractions (top, fraction 1) were analysed by western blotting for myosin Va and rSgII (A). The graph shows the intensity profiles of the two proteins (B). The membranes of fraction 9 of the gradient were pelleted, embedded in `Epon' and analysed by electron microscopy (C). Bar, 500 nm. The arrow indicates the magnified area shown at the bottom right.

View larger version (32K): [in a new window]   Fig. 4. Immunoisolation of SGs. (A) A PNS of PC12 cells was fractionated by differential centrifugation. The fraction enriched in SGs was incubated with myosin Va-specific serum (DIL2), control serum (cs) or without serum (w/o). The membranes bound to antibodies were isolated by immunoprecipitation with magnetic beads covalently coated with secondary antibodies. Bound (bound) and unbound membranes (unbound) were analysed by western blotting using antibodies against SgII. The signals for SgII (SgII) and the IgG heavy chain (IgG) are indicated. (B) The bar graph shows a quantitative analysis of seven independent experiments. For each experiment the sum of SgII in the respective unbound and bound fraction was set to 100%. Note that the differences between values obtained for the myosin Va serum and the controls are highly significant (paired t-test, P<0.005). Error bar, s.e.m.

View larger version (70K): [in a new window]   Fig. 6. Expression of FLAG-MCLT inhibits the cortical localisation of SGs. PC12 cells were either single-transfected with hCgB-GFP(S65T) or double-transfected with hCgB-GFP(S65T) and FLAG or FLAG-MCLT, respectively, incubated for 2 hours at 20°C and then at 37°C as indicated. Cells were fixed, stained with phalloidin-TRITC for F-actin and then analysed by confocal double-fluorescence microscopy. For each cell, 40 optical sections were taken. (A,B) Overlays of single optical sections for hCgB-GFP(S65T) (green) and F-actin (red) from cells double-transfected with hCgB-GFP(S65T) and FLAG (A) or FLAG-MCLT (B), respectively, and fixed after 1 hour of chase. SGs colocalising with F-actin are indicated by arrowheads, non-colocalising SGs are indicated by arrows. Asterisks, nontransfected cells. Bar, 5 µm. (C) Quantitation of colocalisation. The 40 optical sections per colour channel were rendered with IPLab 3D-software to 3D representations (see Materials and Methods) and the percentage of SGs colocalising with F-actin was determined. The graph shows mean values for at least four cells per time point and condition from two independent experiments. n, number of SGs evaluated per condition. Error bars, s.e.m.

View larger version (59K): [in a new window]   Fig. 7. Expression of FLAG-MCLT leads to clusters of SGs that colocalise with FLAG-MCLT. PC12 cells were double-transfected with hCgB-GFP(S65T) and FLAG (A-B' or FLAG-MCLT (C-E'), respectively, incubated for 2 hours at 20°C and then for 0 or 90 minutes at 37°C as indicated. Cells were fixed and immunostained against TGN38 (A-C') or the FLAG epitope (D-E'). For confocal double-fluorescence microscopy, 40 optical sections were taken for each cell. (A-C') Three-dimensional representations showing hCgB-GFP(S65T) in green and TGN38 immunostaining in red. The asterisk in B indicates the TGN of a non-transfected cell. (A-C) top views; (A'-C'), side views. Bar, 5 µm. Note that in the presence of FLAG-MCLT SGs accumulate between the TGN and the juxtaposed PM. (D-E') Single confocal section through the center of the cell (D,D') or overlay of all 40 sections (E,E'). (D,E) FLAG-MCLT-immunostaining. (D',E') Overlay of hCgB-GFP(S65T) (green) and FLAG-MCLT-immunostaining (red). Colocalising signals are shown in yellow. (F) PC12 cells were co-transfected with PTS1-GFP and FLAG-MCLT, incubated for 2 hours at 20°C and then for 90 minutes at 37°C. Thereafter cells were fixed, immunostained against the FLAG epitope and analysed by confocal microscopy. A 3D image was rendered. Green, GFP-fluorescence, red, immunofluorescence. Bar, 5 µm. See corresponding movie at http://jcs.biologists.org/supplemental.

View larger version (30K): [in a new window]   Fig. 8. The role of myosin Va in the transport of secretory granules. (A) After MT-dependent delivery from the TGN to the actin cortex, SGs (green spheres) are captured and removed from the cortical entry site by SG-bound active myosin Va (black circles) via transport along actin fibers as indicated by arrows. (B) The expression of the tail fragment of myosin Va (white semi-circles) blocks myosin Va-mediated capturing and transport of SGs in the actin cortex. This leads to a loss in cortical distribution and formation of clusters of SGs at the interface of the MT- and actin-networks.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter