Kazuyuki Matsushima

Distinct neuronal localization of microtubule-associated protein 4 in the mammalian brain.

Although recent studies have suggested the role of microtubule-associated protein (MAP) 4 in some neuron-specific events, there are no reports that directly observed its neuronal localization. Here we show the detailed expression of MAP4 in the mammalian brain. Immunoblotting revealed the presence of MAP4 in all neuronal tissues. The site-specific localization of MAP4 was observed in sagittal brain sections: MAP4 was rich in brain-specific cells, cerebellum Purkinje cells and hippocampus pyramidal cells. When primary cultures of cortical neurons were immunostained, MAP4 was detected in the cell bodies and processes with patchy staining pattern. These results suggested that MAP4 play some roles in the central nervous system, such as the dynamic cytoskeletal reorganization and regulation of the microtubule-dependent long-range transport.

Differences in the regulation of microtubule stability by the pro-rich region variants of microtubule-associated protein 4.

We have recently reported a neural variant of microtubule‐associated protein 4 with a short pro‐rich region (MAP4‐SP). Here, we show that the neural MAP4 has reduced microtubule‐stabilizing activity, compared to the ubiquitous MAP4 with a long pro‐rich region (MAP4‐LP), both in vitro and in vivo. Fluorescence recovery after photobleaching analyses revealed that the interaction of MAP4‐SP with the microtubules is very rapid, with a half‐time of fluorescence recovery of 7 ± 2.36 s, compared to 19.5 ± 3.03 s in case of MAP4‐LP. The dynamic interaction of MAP4‐SP with microtubules in neural cells may contribute to the dynamic behaviors of extending neurites.

Microtubule-associated protein 4 binds to actin filaments and modulates their properties

We previously reported that an isoform of microtubule-associated protein 4 (MAP4) is localized to the distal area of developing neurites, where microtubules are relatively scarce, raising the possibility that MAP4 interacts with another major cytoskeletal component, actin filaments. In the present study, we examined the in vitro interaction between MAP4 and actin filaments, using bacterially expressed MAP4 and its truncated fragments. Sedimentation assays revealed that MAP4 and its microtubule-binding domain fragments bind to actin filaments under physiological conditions. The apparent dissociation constant and the binding stoichiometry of the fragments to actin were about 0.1 µm and 1 : 3 (MAP4/actin), respectively. Molecular dissection studies revealed that the actin-binding site on MAP4 is situated at the C-terminal part of the proline-rich region, where the microtubule-binding site is also located. Electron microscopy revealed that the MAP4-bound actin filaments become straighter and longer and that the number of actin bundles increases with greater concentrations of added MAP4 fragment, indicating that MAP4 binding alters the properties of the actin filaments. A multiple sequence alignment of the proline-rich regions of MAP4 and tau revealed two putative actin-binding consensus sequences.

Microtubule-Associated Protein 4

In contrast with neural microtubule-associated proteins (MAPs) such as MAP1, MAP2, and tau, MAP4 is identified as a ubiquitous MAP. MAP4 stimulates tubulin polymerization and stabilizes polymerized-microtubules as do neural MAPs. Because MAP2, MAP4, and tau are structurally similar, the three MAPs are considered to constitute a superfamily. The architecture of the superfamily proteins consists of an amino-terminal projection domain and a carboxyl-terminal microtubule-binding domain, and the microtubule-binding domain is further divided into three subdomains: the Pro-rich region, the repeat region, and the tail region. Recent studies have revealed the functions of these domains/subdomains in the MAP4 molecule: the projection domain keeps individual microtubules separated by suppressing the bundle-forming ability of the microtubule-binding domain, the Pro-rich region promotes the nucleation of microtubules, the repeat region promotes their elongation, and the tail region may contribute to the proper folding of the molecule. Isoforms of MAP4 are produced from the single MAP4 gene by alternative RNA splicing, thereby five isoforms, with a deletion in the Pro-rich region or the repeat region, are expressed. The expression of these isoforms depends on the tissue type and the developmental stage, suggesting that the function of MAP4 is elaborately regulated by alternative splicing. In this chapter, we will address the structural and functional futures of MAP4 focusing on the functions of domains/subdomains and add some insights into the roles of the protein in neurons.

The Smallest Active Fragment of Microtubule-Associated Protein 4 and Its Interaction with Microtubules in Phosphate Buffer

To analyze the interaction between microtubule-associated protein (MAP) 4 and microtubules physicochemically, a MAP4 active site fragment was designed for nuclear magnetic resonance (NMR) use. The fragment was bacterially expressed and purified to homogeneity. The buffer conditions for NMR were optimized to support microtubule assembly. The fragment was found to bind to microtubules under the optimized buffer conditions.

Primary cilia-mediated intercellular signaling in hair follicles

Dermal papilla cells (DPCs) play a major role in the physiology of hair follicles. The dynamic hair growth is achieved through the sound intercellular communication. It has been demonstrated that the interactive characteristics of DPCs become more remarkable upon the spheroid formation. However, the molecular basis of the activation has remained unclear. Here we show that the novel function of sensory organelle, i.e., primary cilia, of DPCs in the intercellular interaction with other cells. When the formation of primary cilia was inhibited, the DPC-conditioned media became less effective in the augmenting activity of the cellular proliferation of other mesenchymal stromal cells. In the same condition, it was revealed that the gene expression of FGF10 was down-regulated in DPCs. These results indicate that the primary cilia is involved in the production of the growth factor, which could in turn augment the cell growth. Compared to monolayered cells, spheroidal DPCs showed considerably longer primary cilia. The microsphere accordingly supported the growth of the acceptor cells more efficiently than the 2D cultured cells. However, the proliferation efficiency became lower as the spheroidal size became larger. Interestingly, the appearance frequency of primary cilia was reduced in the larger spheroids while the length of the residual organelle kept unchanged. Collectively, our current findings suggest that DPCs tune both the length and the appearance frequency of primary cilia, through which the cells regulate the production of signaling molecules and thereby communicate with other cells. Abbreviations: DPC: dermal papilla cell; siRNA: small-interfering RNA; DCM: DPC-conditioned medium; siKif3a: Kif3a-siRNA Introduction Dermal papilla cells (DPCs) communicate with epidermal and mesenchymal-derived cells by secreting various intercellular signaling molecules, and thereby the cells play essential roles in hair growth and development [1,2]. The hair-inducing activity is regained by forming spheroidal micro tissues that mimic the physiological structure of dermal papillae [3]. It was demonstrated that the three-dimensional spheroidal DPCs express higher levels of intercellular signaling genes, such as wnt, bmp and fgf, compared to two-dimensional monolayered DPCs [4,5]. A great amount of attention has been paid to the primary cilium as a signaling center in mammalian cells. It is reported that the organelle coordinates molecular signaling through Wnt-, FGFor PDGFinvolved pathways [6]. The primary cilia of dermal cells were shown to be critical for hair follicle morphogenesis in mice: when either Kif3a or Ift88 was knocked-out in the dermal cells, the mice showed severe lack of hair [7]. However, the direct role of the primary cilia of DPCs in intercellular signaling remains to be elucidated. In addition, the role of the organella in the hair growth cycle has not been understood. In this study, by using a small-interfering technology and examining the spheroidal DPCs, we showed that the primary cilia of DPCs are involved in the regulation of intercellular signaling with other cells. Materials and methods Cell culture Human primary DPCs from 2 Caucasian females were purchased from PromoCell (Heidelberg, Germany). The cells were routinely grown in Dulbeccos modified Eagles medium (DMEM) supplemented with 4% fetal calf serum (FCS). 3T3-L1 fibroblasts, a type of mesenchymalderived cell line, were maintained in DMEM supplemented with 10% FCS. Fifty U/ml penicillin and 50 μg/ml streptomycin were added into these media. The cells were kept in a 5% CO2 atmosphere at 37 °C. Formation of spheroidal DPCs DPCs were placed on ultra-low-attachment plates (Corning Inc., New York, USA) at cellular densities of 0.5-3.0 × 104 cells/ well. After 3 days, the resultant spheroids were subjected to either immunofluorescent staining or the determination of the cell number (explained below). Preparation of DPC-conditioned media (DCM) To prepare DCM from the siRNA-treated DPCs, siRNA-lipofection was performed in the section below. DCM was collected after 24 hours of the incubation. DCMs were clarified by centrifugation at 10,000 x g for 3 min before use. Determination of DPC number After harvesting DCM, DPCs were treated with trypsin/EDTA at Correspondence to: Kuniyoshi Kaseda, Saravio Central Institute, Saravio Cosmetics Ltd., 1356-6 Oaza Tsurumi, Beppu, Oita 874-0840, Japan, Tel: +81(0)977-75-8575; Fax: +81-(0)977-75-8112; E-mail: kaseda@saravio.jp