First published online 17 August 2004
doi: 10.1242/jcs.01326
Journal of Cell Science 117, 4537-4549 (2004)
Published by The Company of Biologists 2004
Distribution and functions of kinectin isoforms
Niovi Santama1,*,
Connie P. N. Er2,*,
Lee-Lee Ong2 and
Hanry Yu3,4,
1 University of Cyprus and Cyprus Institute of Neurology and Genetics, PO Box 20537, 1678 Nicosia, Cyprus
2 National University Medical Institutes and Faculty of Medicine, National University of Singapore, Block MD11, #04-01A, Clinical Research Center, 10 Medical Drive, Singapore 117597, Rep. of Singapore
3 Department of Physiology, Faculty of Medicine, National University of Singapore, Block MD11, #04-01A, Clinical Research Center, 10 Medical Drive, Singapore 117597, Rep. of Singapore
4 Institute of Bioengineering and Nanotechnology, Agency for Science, Technology and Research, 31 Biopolis Way, The Nanos, #04-01, Singapore 117586, Rep. of Singapore
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Fig. 3. Constructs harbouring kinectin and kinesin domains. cDNA fragments spanning selected sequences of human kinectin and human ubiquitous kinesin heavy chain are illustrated. They were subcloned as C-terminal fusions to the GFP ORF in vector pEGFP-C1 and used for transient transfections of HeLa cells. `Variable inserts' 1-6 in human kinectin form the `variable' C-terminal domain and their locations are illustrated (see Materials and Methods for details). The kinesin-interacting domain of kinectin is within residues 1188-1288, shared between inserts V3 and V4 (Ong et al., 2000 ).
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Fig. 1. Structure and distributions of kinectin isoforms. (A) Schematic diagram depicting the structure of mouse kinectin cDNA. Grey triangles represent the five insertion sequences (variable domains or `inserts' V1-V5) that are used combinatorially to generate splice variants with alternative C-termini. The five inserts are 69, 87, 72, 84 and 110 bp in size, maintaining the correct ORF. V5 includes a stop codon and generates a C-terminus similar to that described in human and chicken whereas lack of V5 creates a novel, 11 a.a. C-terminus extending past the insertion site. The positions of oligonucleotide pair V0up/V0do, used to co-amplify putative kinectin isoforms from mouse hippocampal tissue of distinct developmental stages and primary mouse astrocyte cultures, is indicated with arrows. (B) RT-PCR co-amplification of kinectin isoforms from E15 embryonic mouse hippocampus, using oligonucleotide pair V0up/V0do. At least five differently sized products are detectable on the gel (arrowheads). The mixed product was subcloned and a large number of clones subsequently analysed (see text). (C) Agarose electrophoresis of PCRs, using oligonucleotide pair V0up/V0do with cDNAs cloned in plasmid pCR2.1. Distinct sets of kinectin isoforms derived from mouse E15 and adult hippocampus and astrocytic cultures, respectively are shown.
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Fig. 2. Kinectin intracellular localisation in PC12 cells as revealed by anti-kinectin antibodies. (A) Staining using anti-CT1, reactive to all kinectin isoforms, reveals reticular cytoplasmic labelling. (B) Staining with anti-insert V2 antibody, reactive to insert V2-containing isoforms, results in cytoplasmic reticular labelling as in A, plus distinct labelling of the tips of neurite-like processes (arrow heads). (C) No labelling occurs using pre-immune serum. (D1-D3) Double immunofluorescence localisation of kinectin isoforms and the ER. (D1) Labelling using anti-insert V3 antibody, reactive to insert V3-bearing isoforms, again results in cytoplasmic reticular staining plus staining of neuritic processes (arrowheads); (D2) anti-PDI antibody to visualise the ER; (D3) overlay of the two images. Bars, 5 µm.
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Fig. 4. Roles of 160 kDa and 120 kDa kinectin in ER and mitochondria dynamics. (A) Localization of 160 kDa and 120 kDa kinectin. (a) HeLa cells, transfected with the GFP-TM construct, showed a distinct ER-like network staining. (b) Transient expression of N-terminally truncated non-TM construct, representing 120 kDa kinectin, showed in contrast, strong organelle-like aggregates and occasionally faint ER-like network labelling (arrow). Bar, 10 µm. (B) Effects of overexpression of TM domain on ER and mitochondria distribution in HeLa cells. ER network was labelled by anti-calreticulin mAb (red). Cells transfected with the TM construct were identified with GFP expression (green). (a) An enrichment of TM-containing protein was observed over the area where ER staining localized. (a') ER network integrity was affected after prolonged expression of the TM construct. Mitochondria were labelled with anti-Mn SOD (red). (b,b') Cells transfected with the TM construct (green) showed normal mitochondrial distribution. Bar, 20 µm.
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Fig. 5. Effects of overexpression of kinectin-kinesin binding domains on ER distribution in HeLa cells. (A) Typical images. Anti-calreticulin mAb was used to label the ER network (red). Transfected cells were identified by GFP expression (green). The network was normally distributed in a and a', representing GFP-only transfected cells (control). A collapsed ER network distribution was observed in KNT+ transfected cells (b,b'). Bar, 20 µm. (B) Quantification. The values shown represent the mean±s.e.m. of three experiments. A total of 90 cells were analysed per construct.
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Fig. 6. Association of 120 kDa kinectin isoform with mitochondria in HeLa cells. (A) Western blot of post-nuclear supernatant (lane 1) and mitochondria-enriched fraction (lane 2) of equal concentrations (7 mg/ml), probed with the mitochondrial marker anti-Mn SOD (25 kDa). (B) CT-1 was used to probe the post-nuclear supernatant (lane 1) and mitochondrial fractions (lane 2) of equal concentration (7 mg/ml). 160 kDa and 120 kDa species were detected in lane 1. Only a 120 kDa species was detected in lane 2. (C) The specificity of the affinity-purified anti-insert V2 and V3 antibodies was tested by immunoblotting of a crude cell lysate (concentration 2 mg/ml) Coomassie Blue-stained lysate (lane 1), purified anti-insert V2 and V3 antibodies recognized the prominent 160 kDa form of kinectin (lanes 2 and 3, respectively). (C') Anti-insert V3 antibody was used to probe the post-nuclear supernatant (lane 1) and mitochondrial fractions (lane 2) of equal concentration (7 mg/ml). 160 kDa and 120 kDa kinectin were detected in lane 1 whereas a 120 kDa band was detected in lane 2. (D) An ER marker, anti-calreticulin (63 kDa), was used to probe the post-nuclear supernatant (lane 1) and mitochondrial fractions (lane 2) of equal concentration (0.76 mg/ml). ER protein was not enriched in lane 2.
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Fig. 7. Effects of overexpression of kinectin-kinesin binding domains on anterograde distribution of mitochondria in HeLa cells after heat shock treatment. (A) Typical images. Mitochondria were labelled by anti-Mn SOD Ab, followed by TRITC-conjugated secondary antibody (red). Transfected cells were identified by GFP expression (green). Normal, radially distributed mitochondria, similar to those observed in non-transfected cells, appeared in GFP transfected cells (a,a'). KNT+ transfected cells showed pronounced clustering of mitochondria at the cell centre (b,b'). Bar, 20 µm. (B) Quantification. The values shown represent the mean±s.e.m of three experiments. A total of 90 cells were analysed per construct.
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Fig. 8. Effects of kinectin RNAi on anterograde distribution of ER and mitochondria in HeLa cells. (A) mRNA levels in untransfected cells (lane 1), vector alone stable cells (lane 2) and kinectin knockdown stable cells (lane 3) were determined by semi-quantitative RT-PCR. (B) 10 µg of whole cell lysates as in (A) subjected to western blotting for kinectin and actin proteins. (C) Silencing of kinectin protein. Untransfected HeLa cells (a1-4) and HeLa cells transfected with pSilencer vector control siRNA (b1-4), or with kinectin siRNA (c1-4). Kinectin knockdown stable cells showed distinct disruption in kinectin (c1), ER (c2) and mitochondria (c3) distribution. By contrast, vector control cells (b1-3) and untransfected cells (a1-3) show normal staining patterns. Each cell type was stained for kinectin (anti-CT-1) (red); ER (anti-calreticulin) (red); mitochondria (anti-Mn SOD) (red) and DNA (DAPI) (blue). Microtubule (anti-ß-tubulin) (red) distribution appeared normal for all cells type (a4-c4). Bars, 20 µm.
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© The Company of Biologists Ltd 2004
