Conventional kinesin KIF5B mediates insulin-stimulated GLUT4 movements on microtubules

Insulin stimulates glucose uptake in muscle and adipose cells by mobilizing intracellular membrane vesicles containing GLUT4 glucose transporter proteins to the plasma membrane. Here we show in live cultured adipocytes that intracellular membranes containing GLUT4-yellow fluorescent protein (YFP) move along tubulin-cyan fluorescent protein-labeled microtubules in response to insulin by a mechanism that is insensitive to the phosphatidylinositol 3 (PI3)-kinase inhibitor wortmannin. Insulin increased by several fold the observed frequencies, but not velocities, of long-range movements of GLUT4-YFP on microtubules, both away from and towards the perinuclear region. Genomics screens show conventional kinesin KIF5B is highly expressed in adipocytes and this kinesin is partially co-localized with perinuclear GLUT4. Dominant-negative mutants of conventional kinesin light chain blocked outward GLUT4 vesicle movements and translocation of exofacial Myc-tagged GLUT4-green fluorescent protein to the plasma membrane in response to insulin. These data reveal that insulin signaling targets the engagement or initiates the movement of GLUT4-containing membranes on microtubules via conventional kinesin through a PI3-kinase-independent mechanism. This insulin signaling pathway regulating KIF5B function appears to be required for GLUT4 translocation to the plasma membrane.     

Subunits interactions in kinesin motors

Kinesins form a large and diverse superfamily of proteins involved in numerous important cellular processes. The majority of them are molecular motors moving along microtubules. Conversion of chemical energy into mechanical work is accomplished in a sequence of events involving both biochemical and conformational alternation of the motor structure called the mechanochemical cycle. Different members of the kinesin superfamily can either perform their function in large groups or act as single molecules. Conventional kinesin, a member of the kinesin-1 subfamily, exemplifies the second type of motor which requires tight coordination of the mechanochemical cycle in two identical subunits to accomplish processive movement toward the microtubule plus end. Recent results strongly support an asymmetric hand-over-hand model of "walking" for this protein. Conformational strain between two subunits at the stage of the cycle where both heads are attached to the microtubule seems to be a major factor in intersubunit coordination, although molecular and kinetic details of this phenomenon are not yet deciphered. We discuss also current knowledge concerning intersubunit coordination in other kinesin subfamilies. Members of the kinesin-3 class use at least three different mechanisms of movement and can translocate in monomeric or dimeric forms. It is not known to what extent intersubunit coordination takes place in Ncd, a dimeric member of the kinesin-14 subfamily which, unlike conventional kinesin, exercises a power-stroke toward the microtubule minus end. Eg5, a member of the kinesin-5 subfamily is a homotetrameric protein with two kinesin-1-like dimeric halves controlled by their relative orientation on two microtubules. It seems that diversity of subunit organization, quaternary structures and cellular functions in the kinesin superfamily are reflected also by the divergent extent and mechanism of intersubunit coordination during kinesin movement along microtubules.     

Novel interactions of fission yeast kinesin 8 revealed through in vivo expression of truncation alleles

Fission yeast expresses two kinesin 8s, klp5+ and klp6+, which are important for diverse cellular functions: mitosis, meiosis, and the maintenance of normal cell morphology. During vegetative growth these motors display complex localization patterns, moving from the cytoplasm during interphase to the kinetochores in early mitosis, the interpolar spindle in anaphase B, and then back into the cytoplasm. We have expressed GFP-tagged alleles of domains from these motors, seeking the signals required for their localizations. The tail of Klp5p localized to the interphase nucleus, more specifically to telomeres. Addition of the neck re-directed this fragment to microtubules in the cytoplasm. Klp6-tail and the neck-tail domains of both motors localized at microtubule ends. Klp6-neck-tail localized to the spindle in early mitosis but to the pole-proximal ends of the spindle in anaphase B. The Klp5-motor and motor-neck localized to microtubules, often causing them to bundle. Over-expression of Klp6-motor or motor-neck resulted in shorter microtubules. These localization patterns were no different when constructs were expressed in strains lacking either or both of the endogenous, full-length proteins. Our results indicate that the localization signals for these kinesins are not derived from simple amino acid sequences but from complex interactions among multiple domains of each motor.     

Structures of kinesin and kinesin-microtubule interactions

Several X-ray crystal structures of kinesin motor domains have recently been solved at high resolution ( approximately 0.2-0.3 nm), in both their monomeric and dimeric states. They show the folding of the polypeptide chain and different arrangements of subunits in the dimer. In addition, cryo-electron microscopy and image reconstruction have revealed microtubules decorated with kinesin at intermediate resolution ( approximately 2 nm), showing the distribution and orientation of kinesin heads on the microtubule surface. The comparison of the X-ray and electron microscopy results yields a model of how monomeric motor domains bind to the microtubule but the binding of dimeric motors, their stoichiometry, or the influence of nucleotides remains a matter of debate.     

Mitotic centromere-associated kinesin (MCAK) mediates paclitaxel resistance

Paclitaxel has powerful anticancer activity, but some tumors are inherently resistant to the drug, whereas others are initially sensitive but acquire resistance during treatment. To deal with this problem, it will be necessary to understand the mechanisms of drug action and resistance. Recent studies indicate that paclitaxel blocks cell division by inhibiting the detachment of microtubules from centrosomes. Here, we demonstrate that mitotic centromere-associated kinesin (MCAK), a kinesin-related protein that destabilizes microtubules, plays an important role in microtubule detachment. Depletion of MCAK altered mitotic spindle morphology, increased the frequency of lagging chromosomes, and inhibited the proliferation of WT CHO cells, confirming that it is an essential protein for cell division. In contrast, MCAK depletion rescued the proliferation of mutant paclitaxel-dependent cell lines that are unable to divide because of defective spindle function resulting from altered α-tubulin or class III β-tubulin overexpression. In concert with the correction of mitotic defects, loss of MCAK reversed an aberrantly high frequency of microtubule detachment in the mutant cells and increased their sensitivity to paclitaxel. The results indicate that MCAK affects cell sensitivity to mitotic inhibitors by modulating the frequency of microtubule detachment, and they demonstrate that changes in a microtubule-interacting protein can reverse the effects of mutant tubulin expression.     

Polarity proteins control ciliogenesis via kinesin motor interactions

BACKGROUND: Cilia are specialized organelles that play a fundamental role in several mammalian processes including left-right axis determination, sperm motility, and photoreceptor maintenance. Mutations in cilia-localized proteins have been linked to human diseases including cystic kidney disease and retinitis pigmentosa. Retinitis pigmentosa can be caused by loss-of-function mutations in the polarity protein Crumbs1 (CRB1), but the exact role of CRB1 in retinal function is unclear. RESULTS: Here we show that CRB3, a CRB1-related protein found in epithelia, is localized to cilia and required for proper cilia formation. We also find that the Crumbs-associated Par3/Par6/aPKC polarity cassette localizes to cilia and regulates ciliogenesis. In addition, there appears to be an important role for the polarity-regulating 14-3-3 proteins in this process. Finally, we can demonstrate association of these polarity proteins with microtubules and the microtubular motor KIF3/Kinesin-II. CONCLUSIONS: Our findings point to a heretofore unappreciated role for polarity proteins in cilia formation and provide a potentially unique insight into the pathogenesis of human kidney and retinal disease.     

Functional anatomy of the kinesin molecule in vivo.

We have developed an assay that allows the functional efficiency of mutant kinesins to be probed in vivo. We show here that the growth rate of the filamentous fungus Neurospora crassa can be used as a sensitive reporter for the ability of mutant kinesins to suppress the phenotype of the kinesin null mutant of Neurospora. Truncation mutants, internal deletion mutants and chimeras, in which homologous domains were exchanged between different fungal kinesins, were generated and transformed into the kinesin-deficient strain. None of the mutations affect motor velocity in vitro, but even minor alterations in the tail domain severely compromise kinesin's performance in vivo. The analysis of these mutants has identified subdomains in the stalk and tail likely to be involved in cargo binding and/or regulation of motor activity. The phenotypes of several mutants strongly suggest that kinesin requires a folded conformation to achieve full functionality in vivo. Folding critically depends on two flexible domains in the stalk that allow an interaction of the tail with the neck/hinge region near the catalytic motor domain. The assay has proven to be a valuable tool in the analysis of kinesin function in vivo and should help to characterize the sites involved in intra- and intermolecular interactions.     

Conventional kinesin holoenzymes are composed of heavy and light chain homodimers

Conventional kinesin is a major microtubule-based motor protein responsible for anterograde transport of various membrane-bounded organelles (MBO) along axons. Structurally, this molecular motor protein is a tetrameric complex composed of two heavy (kinesin-1) chains and two light chain (KLC) subunits. The products of three kinesin-1 (kinesin-1A, -1B, and -1C, formerly KIF5A, -B, and -C) and two KLC (KLC1, KLC2) genes are expressed in mammalian nervous tissue, but the functional significance of this subunit heterogeneity remains unknown. In this work, we examine all possible combinations among conventional kinesin subunits in brain tissue. In sharp contrast with previous reports, immunoprecipitation experiments here demonstrate that conventional kinesin holoenzymes are formed of kinesin-1 homodimers. Similar experiments confirmed previous findings of KLC homodimerization. Additionally, no specificity was found in the interaction between kinesin-1s and KLCs, suggesting the existence of six variant forms of conventional kinesin, as defined by their gene product composition. Subcellular fractionation studies indicate that such variants associate with biochemically different MBOs and further suggest a role of kinesin-1s in the targeting of conventional kinesin holoenzymes to specific MBO cargoes. Taken together, our data address the combination of subunits that characterize endogenous conventional kinesin. Findings on the composition and subunit organization of conventional kinesin as described here provide a molecular basis for the regulation of axonal transport and delivery of selected MBOs to discrete subcellular locations.     

12/15-Lipoxygenase activity mediates inflammatory monocyte/endothelial interactions and atherosclerosis in vivo

We have shown that the 12/15-lipoxygenase (12/15-LO) product 12S-hydroxyeicosatetraenoic acid increases monocyte adhesion to human endothelial cells (EC) in vitro. Recent studies have implicated 12/15-LO in mediating atherosclerosis in mice. We generated transgenic mice on a C57BL/6J (B6) background that modestly overexpressed the murine 12/15-LO gene (designated LOTG). LOTG mice had 2.5-fold elevations in levels of 12S-hydroxyeicosatetraenoic acid and a 2-fold increase in expression of 12/15-LO protein in vivo. These mice developed spontaneous aortic fatty streak lesions on a chow diet. Thus, we examined effects of 12/15-LO expression on early events leading to atherosclerosis in these mice. We found that, under basal unstimulated conditions, LOTG EC bound more monocytes than B6 control EC (18 +/- 2 versus 7 +/- 1 monocytes/field, respectively; p < 0.0001). Inhibition of 12/15-LO activity in LOTG EC using a 12/15-LO ribozyme completely blocked monocyte adhesion in LOTG mice. Thus, 12/15-LO activity is required for monocyte/EC adhesion in the vessel wall. Expression of ICAM-1 in aortic endothelia of LOTG mice was increased severalfold. VCAM-1 expression was not changed. In a series of blocking studies, antibodies to alpha(4) and beta(2) integrins in WEHI monocytes blocked monocyte adhesion to both LOTG and B6 control EC. Inhibition of ICAM-1, VCAM-1, and connecting segment-1 fibronectin in EC significantly reduced adhesion of WEHI monocytes to LOTG EC. In summary, these data indicate that EC from LOTG mice are "pre-activated" to bind monocytes. Monocyte adhesion in LOTG mice is mediated through beta(2) integrin and ICAM-1 interactions as well as through VLA-4 and connecting segment-1 fibronectin/VCAM-1 interactions. Thus, 12/15-LO mediates monocyte/EC interactions in the vessel wall in atherogenesis at least in part through molecular regulation of expression of endothelial adhesion molecules.     

Helix capping interactions stabilize the N-terminus of the kinesin neck coiled-coil

In an effort to understand how specific structural features within the kinesin neck, a region of the heavy chain located between the catalytic core and stalk domains, may contribute to motor processivity (an ability to remain attached to the microtubule filament), we have prepared several synthetic peptides corresponding to the neck region of human conventional kinesin and determined their secondary structure content and stability by CD spectroscopy. Our results show that the coiled-coil dimerization domain within the human kinesin neck region corresponds to residues 337 to 369 in solution, and thus is in excellent agreement with the recent X-ray crystallographic structures of rat brain kinesin. Further, we show that the first and last heptads of this region are absolutely critical for creating the high stability and association of the dimeric structure. Interestingly, addition of the 7 N-terminal neck-linker residues (330-336) to the coiled-coil domain significantly increased its stability (Delta GdnHCl midpoint of 1 M or an increase of approximately 1.5 kcal/mol), indicating that a strong structural link exists between the neck-linker and coiled-coil region. Subsequent high-resolution structural analysis of the residues located at the junction of the neck-linker and coiled-coil revealed the presence of the two helix capping motifs, the capping box (a reciprocal interaction of Thr 336 with Gln 339) and the hydrophobic staple (a hydrophobic packing interaction of Leu 335 with Trp 340). Substitution of Leu 335 and Thr 336 (the capping residues) with Gly completely eliminated the increased stability of the coiled-coil region observed in the presence of the neck-linker residues. Correspondingly, substitution of Trp 340, the first hydrophobic core d position residue of the coiled-coil, with an Ala residue resulted in a greater than expected decrease in stability and helicity of the coiled-coil structure. Subsequent analysis of the X-ray structure and substitution analysis of Lys 341 revealed that Trp 340 makes an important interchain hydrophobic interaction with Lys 341 of the opposite chain. Taken together these results reveal that a set of strong intra- and inter-chain interactions made up of the helix "capping box," "hydrophobic staple," and the newly identified "Leu-Trp-Lys sandwich" motifs stabilize the kinesin neck coiled-coil structure, thus preventing it from fraying and unfolding.     

Chromatin looping mediates boundary element promoter interactions

One facet of the control of gene expression is long-range promoter regulation by distant enhancers. It is an important component of the regulation of genes that control metazoan development and has been appreciated for some time but the molecular mechanisms underlying this regulation have remained poorly understood. A recent study by Cleard and colleagues1 reports the first in vivo evidence of chromatin looping and boundary element promoter interaction. Specifically, they studied the function of a boundary element within the cis-regulatory region of the Abdominal-B (Abd-B) gene of Drosophila melanogaster.     

AMPK mediates autophagy during myocardial ischemia in vivo

We have recently shown that autophagy is induced by ischemia and reperfusion in the mouse heart in vivo. Ischemia stimulates autophagy through an AMP activated protein kinase (AMPK)-dependent mechanism, whereas reperfusion after ischemia stimulates autophagy through a Beclin 1-dependent, but AMPK-independent, mechanism. Autophagy plays distinct roles during ischemia and reperfusion: autophagy may be protective during ischemia, whereas it may be detrimental during reperfusion. We will discuss the role of AMPK in mediating autophagy during myocardial ischemia in vivo.     

C-terminal oligomerization of podocin mediates interallelic interactions

Interallelic interactions of membrane proteins are not taken into account while evaluating the pathogenicity of sequence variants in autosomal recessive disorders. Podocin, a membrane-anchored component of the slit diaphragm, is encoded by NPHS2, the major gene mutated in hereditary podocytopathies. We formerly showed that its R229Q variant is only pathogenic when trans-associated to specific 3′ mutations and suggested the causal role of an abnormal C-terminal dimerization. Here we show by FRET analysis and size exclusion chromatography that podocin oligomerization occurs exclusively through the C-terminal tail (residues 283–382): principally through the first C-terminal helical region (H1, 283–313), which forms a coiled coil as shown by circular dichroism spectroscopy, and through the 332–348 region. We show the principal role of the oligomerization sites in mediating interallelic interactions: while the monomer-forming R286Tfs*17 podocin remains membranous irrespective of the coexpressed podocin variant identity, podocin variants with an intact H1 significantly influence each other's localization (r²?=?0.68, P?=?9.2?×?10^?32). The dominant negative effect resulting in intracellular retention of the pathogenic F344Lfs*4-R229Q heterooligomer occurs in parallel with a reduction in the FRET efficiency, suggesting the causal role of a conformational rearrangement. On the other hand, oligomerization can also promote the membrane localization: it can prevent the endocytosis of F344Lfs*4 or F344* podocin mutants induced by C-terminal truncation. In conclusion, C-terminal oligomerization of podocin can mediate both a dominant negative effect and interallelic complementation. Interallelic interactions of NPHS2 are not restricted to the R229Q variant and have to be considered in compound heterozygous individuals.     

Mini-Mu mediates deletion-inversions in vivo by intra-transposon transposition

We have shown that a mini-Mu can transpose into itself in vivo to generate a circle containing only transposon sequences. This deletion-inversion product, which has previously been observed in vitro, is formed by non-replicative transposition and has directly repeated Mu ends. It therefore cannot undergo further rounds of transposition and retains the two copies of the target sequence duplicated in the event. Thus we have been able to confirm that a mini-Mu can undergo non-replicative reactions in vivo and that these generate a 5 bp target site duplication, as has been shown to occur following replicative transposition and lysogenization with Mu.     

Cardiolipin mediates curcumin interactions with mitochondrial membranes

Curcumin, the main molecular ingredient of the turmeric spice, has been reported to exhibit therapeutic properties for varied diseases and pathological conditions. While curcumin appears to trigger multiple signaling pathways, the precise mechanisms accounting for its therapeutic activity have not been deciphered. Here we show that curcumin exhibits significant interactions with cardiolipin (CL), a lipid exclusively residing in the mitochondrial membrane. Specifically, we found that curcumin affected the structures and dynamics of CL-containing biomimetic and biological mitochondrial membranes. Application of several biophysical techniques reveals the CL-promoted association and internalization of curcumin into lipid bilayers. In parallel, curcumin association with CL containing bilayers increased their fluidity and reduced lipid ordering. These findings suggest that membrane modifications mediated by CL interactions may play a role in the therapeutic functions of curcumin, and that the inner mitochondrial membrane in general might constitute a potential drug target.     

Conformations of kinesin: solution vs. crystal structures and interactions with microtubules

Recently, the molecular structures of monomeric and dimeric kinesin constructs in complex with ADP have been determined by X-ray crystallography (Kull et al. 1996; Kozielski et al. 1997 a; Sack et al. 1997). The “motor” or “head” domains have almost identical conformations in the known crystal structures, yet the kinesin dimer is asymmetric: the orientation of the two heads relative to the coiled-coil formed by their neck regions is different. We used small angle solution scattering of kinesin constructs and microtubules decorated with kinesin in order to find out whether these crystal structures are of relevance for kinesin's structure under natural conditions and for its interaction with microtubules. Our preliminary results indicate that the crystal structures of monomeric and dimeric kinesin are similar to their structures in solution, though in solution the center-of-mass distance between the motor domains of the dimer could be slightly greater. The crystal structure of dimeric kinesin can be interpreted as representing two equivalent conformations. Transitions between these or very similar conformational states may occur in solution. Binding of kinesin to microtubules has conformational effects on both, the kinesin and the microtubule. Solution scattering of kinesin decorated microtubules reveals a peak in intensity that is characteristic for the B-surface lattice and that can be used to monitor the axial repeat of the microtubules under various conditions. In decoration experiments, dimeric kinesin dissociates, at least partly, leading to a stoichiometry of 1:1 (one kinesin head per tubulin dimer; Thormählen et al. 1998 a) in contrast to the stoichiometry of 2:1 reported for dimeric ncd. This discrepancy is possibly due to the effect of steric hindrance between kinesin dimers on adjacent binding sites.     

Liposome/white-cell interactions in vivo

The purpose of this study was to establish whether liposomes administered via the intravenous route promote granulocyte aggregation and pulmonary leucostasis. White cells (labelled with [^IIIln]tropolanate) and positively and negatively charged liposomes (containing entrapped [^99mTc]DTPA) were administered i.v. to rats. The blood clearance and tissue distribution of^IIIIn label was not altered by the administration of liposomes (and vice versa) and it is inferred that on intravenous liposome administration liposome/white-cell interactions are unlikely to compromise lung function.     

An asparaginyl endopeptidase mediates in vivo protein backbone cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.