学 位 記 番 号 理工博 第

氏 名 ゴビンダ

シャルマ

所 属 理工学研究科 生命科学専攻 学 位 の 種 類 博士（理学）

学 位 記 番 号 理工博 第

号 学位授与の日付 平

年

月

日

課程・論文の別 学位規則第４条第

項該当

学 位 論 文 題 名

Lemur kinase 1A (LMTK1A)

の細胞骨格構造との相互作用（英文）

論 文 審 査 委 員 主査 教 授 久永 眞市 委員 教 授 門田 明雄 委員 教 授 岡本 龍史

准教授 安藤 香奈絵

【論文の内容の要旨】

Summary

Neurons communicate with each other through neurites, axons and dendrites.

Neurite formation requires coordinated actions of cytoskeletons and membrane trafficking. Two members of cytoskeletons, microtubules and actin filaments serve as tracks for intracellular membrane traffic depending on regions of neurites, microtubules in the shaft and actin filaments at the tip. Membrane vesicles used for membrane supply to the tip of growing neurites are mainly recycling endosomes, which are controlled by Rab11, a small GTPase. LMTK1A, a novel Ser/Thr kinase expressed abundantly in brains, regulates trafficking of Rab11-positive endosomes in neurites. However LMTK1A does not bind directly with Rab11a, and the mechanism how LMTK1A regulates endosome transport is a question to be answered. In this thesis I investigated the interaction of LMTK1A with cytoskeletons, tracks of vesicle transport.

At first, I searched for the substrates or proteins interacting with LMTK1A by

immunoprecipitation from the mouse brain membrane fraction, which was prepared by

differential centrifugation of brain lysed in absence of detergent. Mass spectroscopy

revealed many proteins, particularly those related to cytoskeletons or membrane

trafficking. Although I examined the direct interaction of several candidates with LMTK1A in cultured cells, however, I could not confirm the binding distinctly, probably due to the difference in sample from brain and from cell culture. Then, I studied the interaction of LMTK1A with cytoskeletons, particularly microtubules and actin filaments using cultured PC-12 and Neuro2A cells. I found that LMTK1A affects microtubule organization in Neuro2A cells. Expression of kinase negative (kn) LMTK1A shifted the microtubules from the center of cell to the peripheral region.

Concomitantly, γ-tubulin changed its position from underneath of the nucleus to the lateral side. However it is not clear how microtubule organization is changed by the expression of kn LMTK1A, one of the reason could be change of γ-tubulin position re lative to nucleus. When γ-tubulin is under nucleus, the astral arrays of microtubules emanating form γ-tubulin containing MTOC, can spread unhindered, However when γ-tubulin is beside nucleus, the microtubules need to go around to nucleus, leading to less amount of microtubules in the central region of the cell. The position of MTOC is important for polarization of cell. The change in its position in response to LMTK1A shows that LMTK1A may have a role in cell differentiation.

Further, I found that LMTK1A regulated the transition of endosomal vesicles from the end of microtubule to the cortical actin-rich region, which would be required for supply of membrane components for growth of neurite. While wild type (wt) LMTK1A inhibited such transition, kn LMTK1A could move over to the cortical actin.

Transition of vesicles from microtubule to actin and changing from microtubule

associated motor to actin based motor is limiting step for the unhindered supply of

components for growth of the neurite. Based on my finding, I would like to propose that

LMTK1A enhances minus end-directed movement of Rab11-positive vesicles by

stimulating dynein-dependent movement and regulates the transition of the tracks from

microtubules to actin filaments by suppressing kinesin-dependent movement.