Takao Nakata

Charcot-Marie-Tooth Disease Type 2A Caused by Mutation in a Microtubule Motor KIF1Bβ

The kinesin superfamily motor protein KIF1B has been shown to transport mitochondria. Here, we describe an isoform of KIF1B, KIF1Bbeta, that is distinct from KIF1B in its cargo binding domain. KIF1B knockout mice die at birth from apnea due to nervous system defects. Death of knockout neurons in culture can be rescued by expression of the beta isoform. The KIF1B heterozygotes have a defect in transporting synaptic vesicle precursors and suffer from progressive muscle weakness similar to human neuropathies. Charcot-Marie-Tooth disease type 2A was previously mapped to an interval containing KIF1B. We show that CMT2A patients contain a loss-of-function mutation in the motor domain of the KIF1B gene. This is clear indication that defects in axonal transport due to a mutated motor protein can underlie human peripheral neuropathy.

The MAP kinase kinase kinase MLK2 co-localizes with activated JNK along microtubules and associates with kinesin superfamily motor KIF3

The MLK (mixed lineage) ser/thr kinases are most closely related to the MAP kinase kinase kinase family. In addition to a kinase domain, MLK1, MLK2 and MLK3 each contain an SH3 domain, a leucine zipper domain and a potential Rac/Cdc42 GTPase‐binding (CRIB) motif. The C‐terminal regions of the proteins are essentially unrelated. Using yeast two‐hybrid analysis and in vitro dot‐blots, we show that MLK2 and MLK3 interact with the activated (GTP‐bound) forms of Rac and Cdc42, with a slight preference for Rac. Transfection of MLK2 into COS cells leads to strong and constitutive activation of the JNK (c‐Jun N–terminal kinase) MAP kinase cascade, but also to activation of ERK (extracellular signal‐regulated kinase) and p38. When expressed in fibroblasts, MLK2 co‐localizes with active, dually phosphorylated JNK1/2 to punctate structures along microtubules. In an attempt to identify proteins that affect the activity and localization of MLK2, we have screened a yeast two‐hybrid cDNA library. MLK2 and MLK3 interact with members of the KIF3 family of kinesin superfamily motor proteins and with KAP3A, the putative targeting component of KIF3 motor complexes, suggesting a potential link between stress activation and motor protein function.

Microtubules provide directional cues for polarized axonal transport through interaction with kinesin motor head

Post-Golgi carriers of various newly synthesized axonal membrane proteins, which possess kinesin (KIF5)-driven highly processive motility, were transported from the TGN directly to axons. We found that KIF5 has a preference to the microtubules in the initial segment of axon. Low dose paclitaxel treatment caused missorting of KIF5, as well as axonal membrane proteins to the tips of dendrites. Microtubules in the initial segment of axons showed a remarkably high affinity to EB1–YFP, which was known to bind the tips of growing microtubules. These findings revealed unique features of the microtubule cytoskeletons in the initial segment, and suggested that they provide directional information for polarized axonal transport.

Synergistic effects of MAP2 and MAP1B knockout in neuronal migration, dendritic outgrowth, and microtubule organization

MAP1B and MAP2 are major members of neuronal microtubule-associated proteins (MAPs). To gain insights into the function of MAP2 in vivo, we generated MAP2-deficient (map2−/−) mice. They developed without any apparent abnormalities, which indicates that MAP2 is dispensable in mouse survival. Because previous reports suggest a functional redundancy among MAPs, we next generated mice lacking both MAP2 and MAP1B to test their possible synergistic functions in vivo. Map2 − /−map1b −/− mice died in their perinatal period. They showed not only fiber tract malformations but also disrupted cortical patterning caused by retarded neuronal migration. In spite of this, their cortical layer maintained an “inside-out” pattern. Detailed observation of primary cultures of hippocampal neurons from map2 −/−map1b −/− mice revealed inhibited microtubule bundling and neurite elongation. In these neurons, synergistic effects caused by the loss of MAP2 and MAP1B were more apparent in dendrites than in axons. The spacing of microtubules was reduced significantly in map2 −/−map1b −/− mice in vitro and in vivo. These results suggest that MAP2 and MAP1B have overlapping functions in neuronal migration and neurite outgrowth by organizing microtubules in developing neurons both for axonal and dendritic morphogenesis but more dominantly for dendritic morphogenesis.

Kinesin Superfamily Protein 2A (KIF2A) Functions in Suppression of Collateral Branch Extension

Through interactions with microtubules, the kinesin superfamily of proteins (KIFs) could have multiple roles in neuronal function and development. During neuronal development, postmitotic neurons develop primary axons extending toward targets, while other collateral branches remain short. Although the process of collateral branching is important for correct wiring of the brain, the mechanisms involved are not well understood. In this study, we analyzed kif2a(-/-) mice, whose brains showed multiple phenotypes, including aberrant axonal branching due to overextension of collateral branches. In kif2a(-/-) growth cones, microtubule-depolymerizing activity decreased. Moreover, many individual microtubules showed abnormal behavior at the kif2a(-/-) cell edge. Based on these results, we propose that KIF2A regulates microtubule dynamics at the growth cone edge by depolymerizing microtubules and that it plays an important role in the suppression of collateral branch extension.

The KIF3 motor transports N-cadherin and organizes the developing neuroepithelium

In the developing brain, the organization of the neuroepithelium is maintained by a critical balance between proliferation and cell–cell adhesion of neural progenitor cells. The molecular mechanisms that underlie this are still largely unknown. Here, through analysis of a conditional knockout mouse for the Kap3 gene, we show that post-Golgi transport of N-cadherin by the KIF3 molecular motor complex is crucial for maintaining this balance. N-cadherin and β-catenin associate with the KIF3 complex by co-immunoprecipitation, and colocalize with KIF3 in cells. Furthermore, in KAP3-deficient cells, the subcellular localization of N-cadherin was disrupted. Taken together, these results suggest a potential tumour-suppressing activity for this molecular motor.

Preferential binding of a kinesin-1 motor to GTP-tubulin–rich microtubules underlies polarized vesicle transport

The high affinity of KIF5 for microtubules rich in GTP-tubulin results in polarized motor protein accumulation at axonal tips in neurons and may underlie polarized vesicle transport.

Visualization of slow axonal transport in vivo

In axons, cytoskeletal constituents move by slow transport. However, it remains controversial whether axonal neurofilaments are dynamic structures in which only subunits are transported or whether filaments assemble in the proximal axon and are transported intact as polymers to the axon terminus. To investigate the form neurofilament proteins take during transport, neurons of transgenic mice lacking axonal neurofilaments were infected with a recombinant adenoviral vector encoding epitope-tagged neurofilament M. Confocal and electron microscopy revealed that the virally encoded neurofilament M was transported in unpolymerized form along axonal microtubules. Thus, neurofilament proteins are probably transported as subunits or small oligomers along microtubules, which are major routes for slow axonal transport.

A complex of GRB2-dynamin binds to tyrosine-phosphorylated insulin receptor substrate-1 after insulin treatment

Insulin drives the formation of a complex between tyrosine‐phosphorylated IRS‐1 and SH2‐containing proteins. The SH2‐containing protein Grb2 also possesses adjacent SH3 domains, which bind the Ras guanine nucleotide exchange factor Sos. In this report, we examined the involvement of another SH3 binding protein, dynamin, in insulin signal transduction. SH3 domains of Grb2 as GST fusion proteins bound dynamin from lysates of CHO cells expressing wild‐type insulin receptor (IR) (CHO‐IR cells) in a cell‐free system (in vitro). Immunoprecipitation studies using specific antibodies against Grb2 revealed that Grb2 was co‐immunoprecipitated with dynamin from unstimulated CHO‐IR cells. After insulin treatment of CHO‐IR cells, anti‐dynamin antibodies co‐immunoprecipitated the IR beta‐subunit and IRS‐1, as tyrosine‐phosphorylated proteins and PI 3‐kinase activity. However, purified rat brain dynamin did not bind directly to either the IR, IRS‐1 or the p85 subunit of PI 3‐kinase in vitro. Together, these results suggest that in CHO‐IR cells, insulin stimulates the binding of dynamin to tyrosine‐phosphorylated IRS‐1 via Grb2 and that IRS‐1 also associates with PI 3‐kinase in response to insulin. This complex formation was reconstituted in vitro using recombinant baculovirus‐expressed IRS‐1, GST‐Grb2 fusion proteins and dynamin peptides containing proline‐rich sequences. Furthermore, dynamin GTPase activity was found to be stimulated when an IRS‐1‐derived phosphopeptide, containing the Grb2 binding site, was added to the dynamin‐Grb2 complex in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

Role of KIFC3 motor protein in Golgi positioning and integration

KIFC3, a microtubule (MT) minus end–directed kinesin superfamily protein, is expressed abundantly and is associated with the Golgi apparatus in adrenocortical cells. We report here that disruption of the kifC3 gene induced fragmentation of the Golgi apparatus when cholesterol was depleted. Analysis of the reassembly process of the Golgi apparatus revealed bidirectional movement of the Golgi fragments in both wild-type and kifC3 −/− cells. However, we observed a markedly reduced inwardly directed motility of the Golgi fragments in cholesterol-depleted kifC3 −/− cells compared with either cholesterol-depleted wild-type cells or cholesterol-replenished kifC3 −/− cells. These results suggest that (a) under the cholesterol-depleted condition, reduced inwardly directed motility of the Golgi apparatus results in the observed Golgi scattering phenotype in kifC3 −/− cells, and (b) cholesterol is necessary for the Golgi fragments to attain sufficient inwardly directed motility by MT minus end–directed motors other than KIFC3, such as dynein, in kifC3 −/− cells. Furthermore, we showed that Golgi scattering was much more drastic in kifC3 −/− cells than in wild-type cells to the exogenous dynamitin expression even in the presence of cholesterol. These results collectively demonstrate that KIFC3 plays a complementary role in Golgi positioning and integration with cytoplasmic dynein.