The two-step cargo recognition mechanism of heterotrimeric kinesin.

Kinesin-driven intracellular transport is essential for various cell biological events and thus plays a crucial role in many pathological processes. However, little is known about the molecular basis of the specific and dynamic cargo-binding mechanism of kinesins. Here, an integrated structural analysis of the KIF3/KAP3 and KIF3/KAP3-APC complexes unveils the mechanism by which KIF3/KAP3 can dynamically grasp APC in a two-step manner, which suggests kinesin-cargo recognition dynamics composed of cargo loading, locking, and release. Our finding is the first demonstration of the two-step cargo recognition and stabilization mechanism of kinesins, which provides novel insights into the intracellular trafficking machinery.

Interaction between Teneurin-2 and microtubules via EB proteins provides a platform for GABAA receptor exocytosis.

Neurons form dense neural circuits by connecting to each other via synapses and exchange information through synaptic receptors to sustain brain activities. Excitatory postsynapses form and mature on spines composed predominantly of actin, while inhibitory synapses are formed directly on the shafts of dendrites where both actin and microtubules (MTs) are present. Thus, it is the accumulation of specific proteins that characterizes inhibitory synapses. In this study, we explored the mechanisms that enable efficient protein accumulation at inhibitory postsynapse. We found that some inhibitory synapses function to recruit the plus end of MTs. One of the synaptic organizers, Teneurin-2 (TEN2), tends to localize to such MT-rich synapses and recruits MTs to inhibitory postsynapses via interaction with MT plus-end tracking proteins EBs. This recruitment mechanism provides a platform for the exocytosis of GABAA receptors. These regulatory mechanisms could lead to a better understanding of the pathogenesis of disorders such as schizophrenia and autism, which are caused by excitatory/inhibitory (E/I) imbalances during synaptogenesis.

Correlative microscopy and block-face imaging (CoMBI): a 3D imaging method with wide applicability in the field of biological science.

Correlative microscopy and block-face imaging (CoMBI) is an imaging method, which is characterized by the ability to obtain both serial block-face images as a 3-dimentional (3D) dataset and sections for 2-dimentional (2D) light microscopic analysis. These 3D and 2D morphological data can be correlated with each other to facilitate data interpretation. CoMBI is an easy-to-install and low-cost 3D imaging method since its system can be assembled by the researcher using a regular microtome, consumer digital camera, and some self-made devices, and its installation and instruction manuals are open-source. After the first release of CoMBI method from our laboratory, CoMBI systems have been installed in more than a dozen laboratories and are used for 3D analysis of various biological specimens. Typical application of CoMBI is 3D anatomical analysis using the natural color and contrast of the specimen. We have been using CoMBI for analyzing human brain to obtain the fine 3D anatomy as a reference to determine the causes of neurological diseases and to improve the effectiveness of surgery. Recently, we have been using CoMBI for detecting the colors of chromogens, which are used for labeling specific molecules. Mouse embryos colored with X-gal, a conventional chromogen for detecting LacZ products, were imaged using CoMBI, and the 3D distribution of X-gal was successfully visualized. Thus, CoMBI can now be used for many purposes, including 3D anatomical analysis, 2D microscopy using sections, and 3D distribution of specific molecules. These suggest that CoMBI should be more widely used in the field of biological research.

Recent technological advances in correlative light and electron microscopy for the comprehensive analysis of neural circuits.

Light microscopy (LM) covers a relatively wide area and is suitable for observing the entire neuronal network. However, resolution of LM is insufficient to identify synapses and determine whether neighboring neurons are connected via synapses. In contrast, the resolution of electron microscopy (EM) is sufficiently high to detect synapses and is useful for identifying neuronal connectivity; however, serial images cannot easily show the entire morphology of neurons, as EM covers a relatively narrow region. Thus, covering a large area requires a large dataset. Furthermore, the three-dimensional (3D) reconstruction of neurons by EM requires considerable time and effort, and the segmentation of neurons is laborious. Correlative light and electron microscopy (CLEM) is an approach for correlating images obtained via LM and EM. Because LM and EM are complementary in terms of compensating for their shortcomings, CLEM is a powerful technique for the comprehensive analysis of neural circuits. This review provides an overview of recent advances in CLEM tools and methods, particularly the fluorescent probes available for CLEM and near-infrared branding technique to match LM and EM images. We also discuss the challenges and limitations associated with contemporary CLEM technologies.

Betaine ameliorates schizophrenic traits by functionally compensating for KIF3-based CRMP2 transport.

In schizophrenia (SCZ), neurons in the brain tend to undergo gross morphological changes, but the related molecular mechanism remains largely elusive. Using Kif3b+/- mice as a model with SCZ-like behaviors, we found that a high-betaine diet can significantly alleviate schizophrenic traits related to neuronal morphogenesis and behaviors. According to a deficiency in the transport of collapsin response mediator protein 2 (CRMP2) by the KIF3 motor, we identified a significant reduction in lamellipodial dynamics in developing Kif3b+/- neurons as a cause of neurite hyperbranching. Betaine administration significantly decreases CRMP2 carbonylation, which enhances the F-actin bundling needed for proper lamellipodial dynamics and microtubule exclusion and may thus functionally compensate for KIF3 deficiency. Because the KIF3 expression levels tend to be downregulated in the human prefrontal cortex of the postmortem brains of SCZ patients, this mechanism may partly participate in human SCZ pathogenesis, which we hypothesize could be alleviated by betaine administration.

The Spatiotemporal Construction of the Axon Initial Segment via KIF3/KAP3/TRIM46 Transport under MARK2 Signaling.

The axon initial segment (AIS) is a compartment that serves as a molecular barrier to achieve axon-dendrite differentiation. Distribution of specific proteins during early neuronal development has been proposed to be critical for AIS construction. However, it remains unknown how these proteins are specifically targeted to the proximal axon within this limited time period. Here, we reveal spatiotemporal regulation driven by the microtubule (MT)-based motor KIF3A/B/KAP3 that transports TRIM46, influenced by a specific MARK2 phosphorylation cascade. In the proximal part of the future axon under low MARK2 activity, the KIF3/KAP3 motor recognizes TRIM46 as cargo and transports it to the future AIS. In contrast, in the somatodendritic area under high MARK2 activity, KAP3 phosphorylated at serine 60 by MARK2 cannot bind with TRIM46 and be transported. This spatiotemporal regulation between KIF3/KAP3 and TRIM46 under specific MARK2 activity underlies the specific transport needed for axonal differentiation.

KIF1Bß mutations detected in hereditary neuropathy impair IGF1R transport and axon growth.

KIF1Bß is a kinesin-3 family anterograde motor protein essential for neuronal development, viability, and function. KIF1Bß mutations have previously been reported in a limited number of pedigrees of Charcot-Marie-Tooth disease type 2A (CMT2A) neuropathy. However, the gene responsible for CMT2A is still controversial, and the mechanism of pathogenesis remains elusive. In this study, we show that the receptor tyrosine kinase IGF1R is a new direct binding partner of KIF1Bß, and its binding and transport is specifically impaired by the Y1087C mutation of KIF1Bß, which we detected in hereditary neuropathic patients. The axonal outgrowth and IGF-I signaling of Kif1b-/- neurons were significantly impaired, consistent with decreased surface IGF1R expression. The complementary capacity of KIF1Bß-Y1087C of these phenotypes was significantly impaired, but the binding capacity to synaptic vesicle precursors was not affected. These data have supported the relevance of KIF1Bß in IGF1R transport, which may give new clue to the neuropathic pathogenesis.

Mechanism of Activity-Dependent Cargo Loading via the Phosphorylation of KIF3A by PKA and CaMKIIa.

A regulated mechanism of cargo loading is crucial for intracellular transport. N-cadherin, a synaptic adhesion molecule that is critical for neuronal function, must be precisely transported to dendritic spines in response to synaptic activity and plasticity. However, the mechanism of activity-dependent cargo loading remains unclear. To elucidate this mechanism, we investigated the activity-dependent transport of N-cadherin via its transporter, KIF3A. First, by comparing KIF3A-bound cargo vesicles with unbound KIF3A, we identified critical KIF3A phosphorylation sites and specific kinases, PKA and CaMKIIa, using quantitative phosphoanalyses. Next, mutagenesis and kinase inhibitor experiments revealed that N-cadherin transport was enhanced via phosphorylation of the KIF3A C terminus, thereby increasing cargo-loading activity. Furthermore, N-cadherin transport was enhanced during homeostatic upregulation of synaptic strength, triggered by chronic inactivation by TTX. We propose the first model of activity-dependent cargo loading, in which phosphorylation of the KIF3A C terminus upregulates the loading and transport of N-cadherin in homeostatic synaptic plasticity.