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doi: 10.1242/10.1242/jcs.00083

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Involvement of conventional kinesin in glucose-stimulated secretory granule movements and exocytosis in clonal pancreatic ß-cells

¹ Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK
² School of Biological Sciences, University of Manchester, 2.205 Stopford Building, Oxford Road, Manchester M13 9PT, UK

View larger version (55K): [in a new window]   Fig. 3. Generation and expression of KHCmut conventional kinesin heavy chain construct. (A) cDNA encoding the rat conventional kinesin heavy chain motor domain containing a T93N point mutation at the ATP-binding consensus motif, and a histidine-tag (6His) was cloned into the XbaI-XhoI sites of pcDNA3.1(-). INS-1 cells were transfected, fixed 48 hours later with cold methanol and then co-immunostained with a rabbit polyclonal anti-6His-tag antibody (1:250) (a) and a mouse monoclonal anti -tubulin antibody (1:1000) (b) and visualised with Alexa 568 and -488 secondary antibodies, respectively. (c) Overlay of a and b. Overlap appears as yellow. (d-f) High magnification of boxed regions in (a-c). Bars, 2.5 µm (a-c); 1.1 µm (d-f). (B) Cells were co-transfected with KHCmut-pAdTrack-CMV and mitochondrial.DsRed. 48 hours after transfection cells were imaged on a confocal microscope. The KHCmut-expressing cells were identified by exciting EGFP at 488 nm and the DsRed fluorescence of the same cells was visualised by exciting at 568 nm. Typical DsRed in vivo confocal images of mitochondria in INS-1 (a,b) and in HeLa cells (c,d) in KHCmut expressing (a,c) and control cells (b,d) are shown. Scattered lines indicate the position of the plasma membrane obtained as an overlay from the transmitted image of the cell. Bars, 2.5 µm (a-d).

View larger version (40K): [in a new window]   Fig. 8. Expression and effect of KHC340 on vesicle movements and secretion. (A) KHC340 construct was generated as KHCmut described under Fig. 3. (B) INS-1 cells were transfected with the KHC340-pcDNA3.1(-), fixed with cold methanol and then co-immunostained with a rabbit polyclonal anti-6His-tag antibody (1:250) (a) and a mouse monoclonal anti -tubulin antibody (1:1000) (b) and visualised with Alexa 568 and -488 secondary antibodies, respectively. (c) Overlay of a and b. Overlap appears as yellow. Bar, 2.5 µm (a-c). (C) Cells were co-transfected with KHC340-pAdTrack-CMV and phogrin.DsRed. Typical 568 nm in vivo confocal images of insulin-containing vesicles (a,c) in KHC340-expressing (a) and control cells (c). Movements of vesicles were imaged and analysed as described in Fig. 5. (b,d) Tracks of granules in a and c. Bars, 2.5 µm (a,c); 1.95 µm (b,d). (D) Effect of KHC340 on glucose-stimulated human growth hormone (hGH) release from MIN6 cells was studied as described in Fig. 7.

View larger version (36K): [in a new window]   Fig. 9. ATP dependence of insulin-containing vesicle movements. (A) Cells transfected with phogrin.EGFP were permeabilised using 20 µM digitonin in intracellular buffer. Vesicle movement was viewed on an Ultra VIEWTM Live Cell Confocal Imaging system (see Materials and Methods). Typical 488 nm in vivo confocal images of insulin-containing vesicles (a,c,e,g) in INS-1 cells are shown. Images were taken every 0.5 seconds for 30 seconds (for a total of 60 frames). The location of vesicles was determined using the image analysis software MethaMorphTM. The movements of vesicles were tracked for 60 frames unless the spot was lost from view. (b,d,f,h) Tracks of granules in a, c, e and g. Bars, 2.5 µm (a,c,e,g); 3.25 µm (b,d,f,h). (B) During imaging the magnification parameters were set at a constant level and the velocity is given in arbitary units. Differences between the behaviour of vesicles at various ATP concentrations were assessed by a 2-test (using Yates's correction) on histograms generated from the distance moved data. The inset shows the probability of vesicles moving >2.8 arbitrary units in the presence of 1.0 mM (filled bar) and 5.0 mM (open bar) ATP. For URL address to movies, see legend to Fig. 5.

View larger version (34K): [in a new window]   Fig. 5. Quantitative analysis of insulin-containing vesicle movements in KHCmut-expressing and control cells. (A) Cells were co-transfected with KHCmut-pAdTrack-CMV and phogrin.DsRed. 48 hours after transfection cells were imaged at a stimulatory glucose concentration (16 mM) on a confocal microscope. The KHCmut-expressing cells were identified by exciting EGFP at 488 nm and the DsRed fluorescence of the same cells was visualised by exciting at 568 nm. Typical 568 nm in vivo confocal images of insulin-containing vesicles (a,c) in KHCmut-expressing (a) and control cells (c) are shown. Images were taken every 5 seconds for 4 minutes or 2 frames/s for 30 seconds (for a total of 60 frames). The movements of vesicles were tracked for 60 frames unless the spot was lost from view. (b,d) Tracks of granules in (a) and (c). Bars, 1 µm (a,c); 0.75 µm (b,d). (B) For quantification of motility, vesicles were randomly selected (20 in each cell) in seven dominant-negative kinesin-expressing (open bars) and seven control cells (closed bars) and the location of granules was determined using the image analysis software MethaMorphTM (see Materials and Methods). The bar diagrams show the probability of vesicles travelling at the indicated velocities. For corresponding movies (Fig. 5Aa,c), see ftp://researcher{at}137.222.66.116/ (long on as `researcher', password c1100cs, directory `kinesin').

View larger version (47K): [in a new window]   Fig. 6. Quantitative analysis of mitochondrial movements in KHCmut-expressing and control cells. Cells were co-transfected with KHCmut-pAdTrack-CMV and mitochondrial.DsRed. Movement of mitochondria was analysed as described in Fig. 5. Typical 568 nm in vivo confocal images of mitochondria (a,c) in KHCmut-expressing (a) and control cells (c). (b,d) Tracks of granules in (a) and (c). Bars, 1 µm (a-d). (B) The bar diagrams show the probability of mitochondria travelling at the indicated velocities. For URL address to movies, see legend to Fig. 5.

View larger version (61K): [in a new window]   Fig. 1. Immunoadsorption of phogrin.EGFP-containing vesicles from INS-1 cell homogenates. Cells were infected with the phogrin.EGFP adenoviral construct and homogenised 24 hours after infection (Hom.). Phogrin.EGFP-containing vesicles were then immunoadsorbed using a polyclonal anti-phogrin antibody (P), a monoclonal anti-EGFP antibody (GFP), or an irrelevant monoclonal anti-sterol response element binding protein 1 (SREBP1) antibody (Cont). The immunoadsorbed vesicles were analysed by 7.5-15% SDS-PAGE and immunoblotting. The blots were probed with: (a) an anti-phogrin antibody; (b) an anti-EGFP antibody; (c) an anti-insulin antibody; (d) an anti-glycerol phosphate dehydrogenase (mGPDH) antibody to detect mitochondrial contamination; or (e) an anti-manose-6-phosphate receptor (M6PR) antibody for identifying lysosomal contamination. Molecular weight markers are indicated on the left and arrows show the position of phogrin.EGFP (a,b).

View larger version (72K): [in a new window]   Fig. 2. Conventional kinesin heavy chain (KHC) is associated with insulin granules in ß-cells. (A) INS-1 cells were homogenised (Hom.) and the phogrin.EGFP-containing vesicles were immunoadsorbed with a monoclonal anti-EGFP antibody (GFP) or with a monoclonal anti-SREBP antibody (Cont.) (for details, see Materials and Methods). Immunoblots were probed with a polyclonal-pan-kinesin antibody (left panel), a rabbit polyclonal anti-ubiquitous conventional KHC antibody (uKHC) (middle panel) or a monoclonal anti-dynein antibody (right panel). 15 µg protein was loaded from the cell homogenates and 2.5 µg from purified kinesin from pig brain (Kin); the protein content of the immunoadsorbed samples was not determined. Note that the immunoreactive band of 60 kDa detected by the anti-dynein antibody in the immunoadsobed sample is corresponds to IgG. (B) INS-1 cells were co-immunostained with a rabbit polyclonal anti-uKHC (1:200) and a guinea pig polyclonal anti-insulin (1:500) antibody and visualised with an Alexa 488 goat anti-rabbit (1:500) and an Alexa 568 goat anti-guinea pig (1:500) secondary antibody. (a) Alexa 488 fluorescence (488 nm excitation). (b) Alexa 568 fluorescence (568 nm excitation). (c) Overlay of a and b. Overlap appears as yellow. (d-f) High magnification of boxed regions in (a-c). Bars, 5 µm (a-c); 0.5 µm (d-f).

View larger version (119K): [in a new window]   Fig. 4. Localisation of insulin and Golgi apparatus in KHCmut-expressing (a-c, g-i; arrow) and control (d-f, g-i) ß-cells. Cells were transfected with the KHCmut-pAdTrack-CMV construct (A). Downstream of the multiple cloning site, the shuttle vector carries cDNA for EGFP driven by a distinct second CMV promoter. 48 hours after transfection the cells were fixed and probed with (a-f) a guinea-pig anti-insulin (1:500) or (g-i) a mouse monoclonal anti-TGN38 antibody (1:100) and then visualised with the appropriate Alexa 568 secondary antibodies. (a,d,g) Transmitted light images. (b,e,h) Alexa 568 fluorescence (568 nm excitation). (c,f,i) Intrinsic EGFP fluorescence (488 nm excitation). Bars, 2.5 µm (a-i).

View larger version (38K): [in a new window]   Fig. 7. Effect of KHCmut on glucose-stimulated human growth hormone (hGH) release from MIN6 and INS-1 cells. Cells were co-transfected with 0.5 µg hGH-encoding plasmid pXGH5 together with 1 µg KHCmut-pAdTrack-CMV or the corresponding empty vector pAdTrack-CMV. (A) For immunocytochemistry, cells were fixed and co-stained with guinea-pig ployclonal anti-insulin (1:500) and mouse monoclonal anti-hGH (1:150) antibodies and then visualised with Alexa 488 goat anti-guinea pig and Alexa 568 goat anti-mouse secondary antibodies. (a) Alexa 488 and (b) Alexa 568 fluorescence (488 and 568 nm excitations, respectively). (c) Overlay of a and b. Overlap appears as yellow. (d-f) High magnification of boxed regions in a-c. Bars, 2.5 µm (a-c); or 0.4 µm (d-f). (B,C) For hGH assay, cells were cultured for 2 days in complete growth medium and then starved in DMEM medium containing 3 mM glucose 12 hours before stimulation. INS-1 (B) or MIN6 (C) cells were incubated first in the presence of 3 mM glucose for 20 minutes and then at 16 mM or 30 mM glucose for 20 or 90 minutes. Following stimulation, the cells were lysed in 0.5% Triton X-100 and assayed for total hGH using a colorimetric sandwich ELISA method. hGH release was expressed as a percentage of the total hGH and was compared with values obtained with the empty vector (pAdTrack-CMV) at 3 mM glucose. Number of transfections for each condition are indicated in the columns. **P<0.001 compared with basal (empty vector at 3 mM glucose).

View larger version (17K): [in a new window]   Fig. 10. Role of kinesin-mediated vesicle translocation in glucose-stimulated secretion. Typical biphasic insulin secretion from pancreatic islet ß-cells when glucose concentration is elevated to 16 mM (upper panel) and the corresponding glucose-stimulated vesicle movements (lower panel) are shown. (1) At sub-stimulatory [glucose], a small proportion (5% of total) (Rorsman et al., 2000) of granules is docked and immediately available for release (releasable pool). However, most of the granules are situated some distance from the plasma membrane in a `reserve pool' and need to be chemically modified or physically translocated to release sites. (2) Shortly after elevation of [glucose], granules that are already docked undergo exocytosis and transient stimulation of insulin secretion is observed. (3) Kinesin-dependent vesicle recruitment. Note that the nadir in the rate of secretion between end of exocytosis of vesicles in the releaseable pool (2), and the activation of kinesin-dependent vesicle recruitment (3) is postulated to be longer in islets than cell lines, where the two phases of secretion are not readily distinguished. (4) ATP-dependent mobilisation of granules from the reserve pool via kinesin-mediated transport. This stage represents the sustained release of insulin. (5) KHCmut blocks the transport of granules from the reverse pool by irreversibly binding to microtubules (Nakata and Hirokawa, 1995; Krylyshkina et al., 2002) and inhibits the second phase of insulin release. KHC, kinesin heavy chain; KHCmut, motor domain of kinesin heavy chain containing a T93N point mutation; KLC, kinesin light chain; MT, microtubule.

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