Kinesin-1 and mitochondrial motility control by discrimination of structurally equivalent but distinct subdomains in Ran-GTP-binding domains of Ran-binding protein 2

The pleckstrin homology (PH) domain is a versatile fold that mediates a variety of protein–protein and protein–phosphatidylinositol lipid interactions. The Ran-binding protein 2 (RanBP2) contains four interspersed Ran GTPase-binding domains (RBD_{n = 1–4}) with close structural homology to the PH domain of Bruton''s tyrosine kinase. The RBD₂, kinesin-binding domain (KBD) and RBD₃ comprise a tripartite domain (R₂KR₃) of RanBP2 that causes the unfolding, microtubule binding and biphasic activation of kinesin-1, a crucial anterograde motor of mitochondrial motility. However, the interplay between Ran GTPase and R₂KR₃ of RanBP2 in kinesin-1 activation and mitochondrial motility is elusive. We use structure–function, biochemical, kinetic and cell-based assays with time-lapse live-cell microscopy of over 260 000 mitochondrial-motility-related events to find mutually exclusive subdomains in RBD₂ and RBD₃ towards Ran GTPase binding, kinesin-1 activation and mitochondrial motility regulation. The RBD₂ and RBD₃ exhibit Ran-GTP-independent, subdomain and stereochemical-dependent discrimination on the biphasic kinetics of kinesin-1 activation or regulation of mitochondrial motility. Further, KBD alone and R₂KR₃ stimulate and suppress, respectively, multiple biophysical parameters of mitochondrial motility. The regulation of the bidirectional transport of mitochondria by either KBD or R₂KR₃ is highly coordinated, because their kinetic effects are accompanied always by changes in mitochondrial motile events of either transport polarity. These studies uncover novel roles in Ran GTPase-independent subdomains of RBD₂ and RBD₃, and KBD of RanBP2, that confer antagonizing and multi-modal mechanisms of kinesin-1 activation and regulation of mitochondrial motility. These findings open new venues towards the pharmacological harnessing of cooperative and competitive mechanisms regulating kinesins, RanBP2 or mitochondrial motility in disparate human disorders.     

Ndel1 palmitoylation: a new mean to regulate cytoplasmic dynein activity

Regulated activity of the retrograde molecular motor, cytoplasmic dynein, is crucial for multiple biological activities, and failure to regulate this activity can result in neuronal migration retardation or neuronal degeneration. The activity of dynein is controlled by the LIS1–Ndel1–Nde1 protein complex that participates in intracellular transport, mitosis, and neuronal migration. These biological processes are subject to tight multilevel modes of regulation. Palmitoylation is a reversible posttranslational lipid modification, which can dynamically regulate protein trafficking. We found that both Ndel1 and Nde1 undergo palmitoylation in vivo and in transfected cells by specific palmitoylation enzymes. Unpalmitoylated Ndel1 interacts better with dynein, whereas the interaction between Nde1 and cytoplasmic dynein is unaffected by palmitoylation. Furthermore, palmitoylated Ndel1 reduced cytoplasmic dynein activity as judged by Golgi distribution, VSVG and short microtubule trafficking, transport of endogenous Ndel1 and LIS1 from neurite tips to the cell body, retrograde trafficking of dynein puncta, and neuronal migration. Our findings indicate, to the best of our knowledge, for the first time that Ndel1 palmitoylation is a new mean for fine‐tuning the activity of the retrograde motor cytoplasmic dynein.     

LIS1 and NDEL1 coordinate the plus-end-directed transport of cytoplasmic dynein

LIS1 was first identified as a gene mutated in human classical lissencephaly sequence. LIS1 is required for dynein activity, but the underlying mechanism is poorly understood. Here, we demonstrate that LIS1 suppresses the motility of cytoplasmic dynein on microtubules (MTs), whereas NDEL1 releases the blocking effect of LIS1 on cytoplasmic dynein. We demonstrate that LIS1, cytoplasmic dynein and MT fragments co-migrate anterogradely. When LIS1 function was suppressed by a blocking antibody, anterograde movement of cytoplasmic dynein was severely impaired. Immunoprecipitation assay indicated that cytoplasmic dynein forms a complex with LIS1, tubulins and kinesin-1. In contrast, immunoabsorption of LIS1 resulted in disappearance of co-precipitated tubulins and kinesin. Thus, we propose a novel model of the regulation of cytoplasmic dynein by LIS1, in which LIS1 mediates anterograde transport of cytoplasmic dynein to the plus end of cytoskeletal MTs as a dynein-LIS1 complex on transportable MTs, which is a possibility supported by our data.     

Dynamin-1-like protein (Dnm1L) interaction with kinesin light chain 1 (KLC1) through the tetratricopeptide repeat (TPR) domains

Kinesin light chain 1 (KLC1) mediates binding of KIF5 motor to specific cargo. Using the yeast two-hybrid screening, we found that mitochondrial fission protein dynamin-1-like protein (Dnm1L) interacted with KLC1, but not KIF5. Dnm1L and KLC1 were co-localized in cultured cells. These results suggest that KLC1 may play a potential role in post-fission mitochondrial transport.     

mNUDC is required for plus‐end‐directed transport of cytoplasmic dynein and dynactins by kinesin‐1

Lissencephaly is a devastating neurological disorder caused by defective neuronal migration. The LIS1 (or PAFAH1B1) gene was identified as the gene mutated in lissencephaly patients, and was found to regulate cytoplasmic dynein function and localization. In particular, LIS1 is essential for anterograde transport of cytoplasmic dynein as a part of the cytoplasmic dynein–LIS1–microtubule complex in a kinesin‐1‐dependent manner. However, the underlying mechanism by which a cytoplasmic dynein–LIS1–microtubule complex binds kinesin‐1 is unknown. Here, we report that mNUDC (mammalian NUDC) interacts with kinesin‐1 and is required for the anterograde transport of a cytoplasmic dynein complex by kinesin‐1. mNUDC is also required for anterograde transport of a dynactin‐containing complex. Inhibition of mNUDC severely suppressed anterograde transport of distinct cytoplasmic dynein and dynactin complexes, whereas motility of kinesin‐1 remained intact. Reconstruction experiments clearly demonstrated that mNUDC mediates the interaction of the dynein or dynactin complex with kinesin‐1 and supports their transport by kinesin‐1. Our findings have uncovered an essential role of mNUDC for anterograde transport of dynein and dynactin by kinesin‐1.     

No Conventional Function for the Conventional Kinesin?

A paper by DeGiorgis et al. (DeGiorgis JA, Petukhova TA, Evans TA, Reese TS. Kinesin-3 is an organelle motor in the squid giant axon. Traffic 2008; DOI: 10.1111/j.1600-0854.2008.00809.x) in this issue of Traffic reports on the identification and function of a second squid kinesin, a kinesin-3 motor. As expected, the newly discovered motor associates with axoplasmic organelles in situ and powers motility along microtubules of vesicles isolated from squid axoplasm. Less expected was the finding that kinesin-3 may be the predominant motor for anterograde organelle movement in the squid axon, which challenges the so far undisputed view that this function is fulfilled by the conventional kinesin, kinesin-1. These novel findings let us wonder what the real function of kinesin-1--the most abundant motor in squid axons--actually is.     

The LIS1-related NUDF protein of Aspergillus nidulans interacts with the coiled-coil domain of the NUDE/RO11 protein

The nudF gene of the filamentous fungus Aspergillus nidulans acts in the cytoplasmic dynein/dynactin pathway and is required for distribution of nuclei. NUDF protein, the product of the nudF gene, displays 42% sequence identity with the human protein LIS1 required for neuronal migration. Haploinsufficiency of the LIS1 gene causes a malformation of the human brain known as lissencephaly. We screened for multicopy suppressors of a mutation in the nudF gene. The product of the nudE gene isolated in the screen, NUDE, is a homologue of the nuclear distribution protein RO11 of Neurospora crassa. The highly conserved NH(2)-terminal coiled-coil domain of the NUDE protein suffices for protein function when overexpressed. A similar coiled-coil domain is present in several putative human proteins and in the mitotic phosphoprotein 43 (MP43) of X. laevis. NUDF protein interacts with the Aspergillus NUDE coiled-coil in a yeast two-hybrid system, while human LIS1 interacts with the human homologue of the NUDE/RO11 coiled-coil and also the Xenopus MP43 coiled-coil. In addition, NUDF coprecipitates with an epitope-tagged NUDE. The fact that NUDF and LIS1 interact with the same protein domain strengthens the notion that these two proteins are functionally related.     

Coupling viruses to dynein and kinesin-1

It is now clear that transport on microtubules by dynein and kinesin family motors has an important if not critical role in the replication and spread of many different viruses. Understanding how viruses hijack dynein and kinesin motors using a limited repertoire of proteins offers a great opportunity to determine the molecular basis of motor recruitment. In this review, we discuss the interactions of dynein and kinesin-1 with adenovirus, the α herpes viruses: herpes simplex virus (HSV1) and pseudorabies virus (PrV), human immunodeficiency virus type 1 (HIV-1) and vaccinia virus. We highlight where the molecular links to these opposite polarity motors have been defined and discuss the difficulties associated with identifying viral binding partners where the basis of motor recruitment remains to be established. Ultimately, studying microtubule-based motility of viruses promises to answer fundamental questions as to how the activity and recruitment of the dynein and kinesin-1 motors are coordinated and regulated during bi-directional transport.     

Mutations in the Voltage-Dependent Anion Channel of the Mitochondrial Outer Membrane Cause a Dominant Nonlethal Growth Impairment

Point mutations at K234 and K236 in the yeast voltage-dependent anionchannel 1 (VDAC1) of the mitochondrial outer membrane have been shown tomarkedly impair the membrane insertion of this protein (Smith etal., 1995; Angeles et al., 1998). Mutants of this type wereexpressed in vivo in a strain of yeast with a disruption in theVDAC1 gene. Expression of the various VDAC1 forms was under the control of aGal1 promoter. Wild-type VDAC1 expression fully complemented the slow growthphenotype caused by the disruption. VDAC1 mutants in which K234 and K236 werereplaced by arginine, glutamate, or glutamine caused a more severe negativeeffect on growth. This effect appeared to be dominant since the mutant VDAC1forms suppressed growth in a yeast strain that retained its VDAC1 gene. Thisapparent dominant negative effect on growth did not seem to be specific forany stage of the cell cycle. However, the growth defect was not lethal as theaffected cells still could accumulate the vital stain, FUN1. Expression of amutant in which K234 had been replaced by glutamate had more serious negativegrowth effects than did a similar mutation at K236. Expression of71-116 VDAC1 complemented the VDAC1 disruption; however, expression ofthe same deletion mutant in which the lysines corresponding to K234 and K236were mutated to glutamate severely impaired growth. These results have shownthat a deficiency of lysine at positions 234 and 236 in VDAC1 causes anonlethal growth defect that is more severe than deletion of 45 amino acidsfrom VDAC1 or disruption of the VDAC1 gene. They also indicate that there is ahierarchy in the importance of these lysines with mutations at K234 being themore serious.     

The LIS1/NDE1 Complex Is Essential for FGF Signaling by Regulating FGF Receptor Intracellular Trafficking

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Role of dynein, dynactin, and CLIP-170 interactions in LIS1 kinetochore function

Mutations in the human LIS1 gene cause type I lissencephaly, a severe brain developmental disease involving gross disorganization of cortical neurons. In lower eukaryotes, LIS1 participates in cytoplasmic dynein-mediated nuclear migration. We previously reported that mammalian LIS1 functions in cell division and coimmunoprecipitates with cytoplasmic dynein and dynactin. We also localized LIS1 to the cell cortex and kinetochores of mitotic cells, known sites of dynein action. We now find that the COOH-terminal WD repeat region of LIS1 is sufficient for kinetochore targeting. Overexpression of this domain or full-length LIS1 displaces CLIP-170 from this site without affecting dynein and other kinetochore markers. The NH2-terminal self-association domain of LIS1 displaces endogenous LIS1 from the kinetochore, with no effect on CLIP-170, dynein, and dynactin. Displacement of the latter proteins by dynamitin overexpression, however, removes LIS1, suggesting that LIS1 binds to the kinetochore through the motor protein complexes and may interact with them directly. We find that of 12 distinct dynein and dynactin subunits, the dynein heavy and intermediate chains, as well as dynamitin, interact with the WD repeat region of LIS1 in coexpression/coimmunoprecipitation and two-hybrid assays. Within the heavy chain, interactions are with the first AAA repeat, a site strongly implicated in motor function, and the NH2-terminal cargo-binding region. Together, our data suggest a novel role for LIS1 in mediating CLIP-170-dynein interactions and in coordinating dynein cargo-binding and motor activities.