|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Cell Biology International (2006) 30, 885894 (Printed in Great Britain)
Kinectin participates in microtubule-dependent hormone secretion in pancreatic islet β-cells
Ji‑Zhong Bai, Yu Mon and Geoffrey W. Krissansen*
Department of Molecular Medicine & Pathology, Faculty of Medical and Health Sciences, University of Auckland, Auckland, New Zealand
Kinectin (KNT) is a candidate membrane receptor for kinesin in the movement of intracellular organelles along microtubules. Isoforms of KNT exist containing different combinations of six small (residues 23–33) variable domains (vd) vd1–6 within the C-terminus. Here we investigate a role for KNT and its isoform KNTvd4− in the transport of amylin and insulin-containing secretory vesicles in the pancreatic islet β-cell line RINm5F. KNTvd4− lacks vd4 that forms the kinesin-binding domain, and hence its role in the cell is an enigma. We report that amylin-containing vesicles also contained insulin, and exhibited microtubule, and small G-protein-dependent secretion. Knockdown of KNT by small interference RNA (siRNA) inhibited amylin expression and secretion. In contrast, recombinant KNTvd4− overexpressed in RINm5F cells associated with amylin-containing vesicles and inhibited amylin secretion, but had no discernible affect on amylin expression. The data suggests that both KNT and KNTvd4− participate in microtubule-dependent secretion of amylin in islet β-cells.
Keywords: Kinectin, Intracellular transport, Amylin, Hormone secretion, Islet β-cells.
*Corresponding author. Tel.: +64 9 373 7599x86280; fax: +64 9 373 7492.
Kinectin (KNT) is a 160
An antibody against the cytoplasmic domain of KNT blocked kinesin-binding to microsomes, and inhibited kinesin-driven plus-end, and dynein-powered minus-end directed vesicle motility (Kumar et al., 1995). KNT progressively accumulates on maturing phagosomes that display high motility, and whose motility is bi-directionally inhibited by KNT fragments derived from the central stalk domain, but not the C-terminus of chicken KNT (Blocker et al., 1997). It interacts with the cargo-binding tail of kinesin at its head-binding and myosin-Va-binding domains via its C-terminus (residues 1188–1288) (Ong et al., 2000). The kinesin-binding domain of KNT enhances microtubule-stimulated kinesin ATPase activity, and over-expression of this domain disrupts kinesin-dependent lysosome transport (Ong et al., 2000). KNT mediates microtubule-dependent RhoG activity by a direct interaction of RhoG with its central domain (Vignal et al., 2001), whereas the C-terminus of KNT (residues 630–935) interacts with small G-proteins including RhoA, Rad and Cdc42 (Hotta et al., 1996).
KNT exists as two protein species of 160 and 120-kDa on SDS-PAGE (Kumar et al., 1998; Vignal et al., 2001). The 120
Here we address the role of KNT, and a kinesin-binding domain deficient (vd4-deficient) KNT isoform, in the trafficking of amylin-containing secretory vesicles in a pancreatic islet β-cell line. While kinesin has been shown to play an essential role in insulin secretion (Meng et al., 1997; Varadi et al., 2002a,b), we report here for the first time that KNT is associated with both amylin and insulin-containing vesicles, and plays an essential role in mediating hormone secretion.
2 Materials and methods
2.1 Antibodies and plasmids
Living colour pER1S2/EGFP cDNAs encoding enhanced green fluorescent protein (GFP), and its derivative cyan fluorescent protein (CFP) were obtained from ClonTech Laboratories (Palo Alto, CA). Expression vectors, pcDNA3.1/Neo(+) and pcDNA3.1/Bsd(+), and a rabbit polyclonal anti-GFP antibody (Living Colour A. v. Peptide) were purchased from ClonTech Laboratories. Peroxidase-conjugated donkey anti-rabbit IgG, and a mouse monoclonal anti-α-tubulin antibody were obtained from Sigma (Poole, UK). A guinea pig polyclonal anti-insulin antibody was purchased from CALTAG Laboratories (Burlingame, CA). TRITC-conjugated sheep anti-guinea pig IgG, and Alexa Fluor 594 donkey anti-mouse IgG were from Molecular Probes (Eugene, USA). A rabbit polyclonal antibody raised against a λ lysogen expressing nucleotides 1378–3304 of human KNT clone L25616 has been described previously (Print et al., 1994).
2.2 Construction of expression plasmids
PCR primers used in the construction of plasmids are detailed in Table 1. To construct an expression vector encoding KNTvd4
PCR primers used in the construction of expression plasmids
To generate the amylin
2.3 Generation of stable amylin
Insulin-secreting RINm5F cells (passage number 30–45) were cultured at 37
2.4 Disruption of the cytoskeleton
Transfectants were incubated in 10 or 20
2.5 RT-PCR analysis of expression of KNT isoforms
Total cellular RNA was isolated from RINm5F islet β-cell cultures using the RNeasy Mini kit from Qiagen (Valencia, CA). RT-PCR was performed using the SuperScript™ III one-step RT-PCR system (Invitrogen Life Technologies Inc., CA, USA). The oligonucleotide primers vd2f 5′ ctctgca atgagttaga gtctttgaag 3′ and vd4r 5′ gccttg tgcaaatcat cagctacctt ct 3′ were designed to bracket vd2 and vd4 of rat KNT. The RT-PCR products were analysed on a 2.5% agarose gel, and sequenced.
2.6 Western blot analysis
Transfectants were lysed for 30
To detect the effect that cytoskeletal disruptors have on secretion of amylin
For immunofluorescence assays, cells seeded overnight on coverglasses in RPMI supplemented with 10% FCS,
2.8 Confocal microscopy
Expression profiles of transfectants were analysed using a Leica TCS SP2 confocal microscope (Leica, Heidelberg, Germany) equipped with high numerical aperture lenses (63
2.9 siRNA knockdown
The siRNA oligonucleotides siKNT (5′-AATGGAAGCAGAGATAGCTCATT-3′), and siGFP (5′-GGCUACGUCCAGGAGCGCACC-3′) were purchased from Dharmacon (Lafayette, CO). Transfectants singly expressing amylin
3.1 Visualization of the amylin secretion pathway
Rat amylin cDNA was C-terminally fused to an enhanced green fluorescent protein (GFP) tag (Fig. 1A), and stably transfected into rat pancreatic RINm5F islet β-cells. Amylin
Schematic representation of rat amylin
Nocodazole, cytochalasin D, which disassembles filamentous actin, and lovastatin, which inhibits small G-proteins, were examined for their effects on the secretion of amylin. As determined by Western blot analysis, treatment of cells with 10 and 20
Cytoskeletal modulators inhibit amylin
3.2 RNA interference of endogenous KNT expression inhibits amylin expression and secretion
Knockdown by RNA interference (RNAi) was used to silence KNT expression in RINm5F cells in order to determine whether KNT is critical for amylin secretion. A synthetic 21-nucleotide siRNA duplex (siKNT) with symmetrical 2-nt 3′ overhangs was prepared against the region encompassing nt 1477–1500 of rat KNT (GenBank accession # XM-341305). siKNT binds to a region just upstream of vd1, and hence would be expected to knockdown all KNT isoforms. A control siRNA (siGFP) against nt 174–194 of GFP was used to target the GFP tag in amylin. The siRNA duplexes were transfected into islet β-cells singly transfected to express amylin
Knockdown of KNT expression by siRNA duplexes inhibits amylin
3.3 RINm5F cells express KNT isoforms lacking the kinesin-binding domain
To establish that kinesin binding-domain-deficient forms of KNT play a role in amylin secretion in RINm5F cells, it was essential to first demonstrate whether such forms were naturally present in RINm5F cells. PCR primers flanking vd2 and vd4 were used to RT-PCR KNT transcripts from total cellular RNA in order to determine whether isoforms lacking vd3 and/or vd4, and were expressed in RINm5F β-cells. Gel analysis (Fig. 5A) followed by sequencing of the PCR products revealed the presence of two vd4-deficient isomers, and two vd4-replete isomers (Fig. 5B). Both the former isoforms contained vd3, but one lacked vd2 in addition to vd4. Thus, kinesin binding-domain-deficient (vd4-deficient) forms of KNT are present in RINm5F cells.
A KNT isoform lacking vd2 and vd4 is expressed in RINm5F cells. A region encoding the C-terminal variable domains 2 and 4 of KNT was amplified by RT-PCR from total RNA of RINm5F cells to identify KNT isomers expressed in RINm5F cells. (A) Agarose gel analysis of RT-PCR products obtained using primers that flank vd2 and 4 (lane 2). Lane 1, 100
3.4 A vd4-deficient KNT isoform associates with amylin
The above experiments demonstrated that KNT influences the expression and secretion of amylin, but the role(s) of kinesin binding-domain-deficient (vd4-deficient) forms of KNT in this secretory process are an enigma. In order to investigate a role for the latter forms, the human KNT isoform (clone L25616) lacking vd2 and vd4 (Print et al., 1994) was fused to a cyan fluorescent protein (CFP) tag generating the protein designated KNTvd4
Overexpressed recombinant KNTvd4
Here we confirmed that both KNT and kinesin binding-deficient isoforms of KNT were endogenously expressed in RINm5F cells. Silencing of KNT expression inhibited amylin
While the above considerations propose a role for KNT in the transport and secretion of amylin-containing vesicles in islet β-cells, we cannot rule out the possibility that KNT is also involved in the synthesis of amylin. Indeed, silencing of KNT led to significantly decreased levels of amylin
Santama et al. (2004) reported that 12 of 16 isoforms identified in the mouse nervous system lacked the whole or part of the kinesin-binding domain, and the only ubiquitous KNT isoform was that lacking all the C-terminal variable inserts. The relative abundance and wide-spread distribution of vd4-deficient forms of KNT is interesting given that such isoforms would not be expected to bind to kinesin. To date there has been no attempt to understand the functions of specific KNT isoforms. We have shown that transcripts encoding vd4-deficient forms of KNT are expressed in RINm5F cells. Overexpressed recombinant KNTvd4− was localized to amylin/insulin secretory vesicles, but was not detectable on the ER where KNT has often previously been localized (Futterer et al., 1995; Toyoshima et al., 1992). Overexpressed KNTvd4− inhibited sustained insulin secretion, but unlike the effect obtained with silencing of KNT expression, it did not influence the cellular levels of amylin
KNT has been predicted to form a dimer and there is a twofold molar excess of KNT over kinesin in unextracted vesicles (Kumar et al., 1998). It is thus plausible that two molecules of vd4-replete KNT are required to form a functional bond with kinesin. In this case, vd4-deficient isoforms of KNT might dimerize with vd4-replete isoforms and thereby antagonize the kinesin-binding function of KNT. The pairing of different isoforms with one another would considerably expand the potential number of different isoforms from the predicted 32 theoretically possible zero- to five-insert permutations (Santama et al., 2004). Each isoform pair may exhibit different cellular distributions and functions, and pairing provides a mechanism by which one isoform could antagonize or enhance the effects of another. Since overexpressed KNTvd4− and silencing of KNT expression had different outcomes in terms of amylin expression and vesicle distribution, it would seem that KNTvd4− does not act simply as a dominant-negative inhibitor of KNT.
The above explanations for KNTvd4−-mediated inhibition of amylin secretion are predicated on the demonstration that kinesin mediates the secretion of amylin. Should this not necessarily be the case, then KNTvd4− itself may mediate amylin secretion by binding other motor protein partners through binding domains residing outside the conventional kinesin-binding motif. Overexpressed KNTvd4− could overwhelm the secretory system, and thereby hinder amylin secretion.
In conclusion, our findings demonstrate that KNT is essential for amylin secretion in pancreatic islet β-cells. The KNTvd4− isoform selectively associates with secretory vesicles, and regulates amylin secretion via a mechanism that appears to differ from KNT-mediated secretion of amylin. These results provide additional insights into the fascinating pathways by which cargoes destined for secretion are transported inside the living cell.
We thank the staff of the Biomedical Imaging Unit, Faculty of Medical and Health Sciences, University of Auckland, for their technical assistance. We are grateful to Dr Peter Barling for his critical review of the manuscript. This work was supported in part by grants from the Marsden Fund, the Health Research Council of New Zealand, the Royal Society of New Zealand, and the Wellcome Trust, UK. We are grateful to the Lottery Grants Board, and the Freemasons (Lodge Discovery) for the supply of equipment.
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Received 20 September 2005/22 April 2006; accepted 2 June 2006doi:10.1016/j.cellbi.2006.06.008