@article {69224, title = {CSPP1 stabilizes microtubules by capping both plus and minus ends.}, journal = {Journal of Molecular Cell Biology}, year = {2024}, month = {2024 Feb 22}, abstract = {

Although the dynamic instability of microtubules (MTs) is fundamental to many cellular functions, quiescent MTs with unattached free distal ends are commonly present and play important roles in various events to power cellular dynamics. However, how these free MT tips are stabilized remains poorly understood. Here, we report that centrosome and spindle pole protein 1 (CSPP1) caps and stabilizes both plus and minus ends of static MTs. Real-time imaging of laser-ablated MTs in live cells showed deposition of CSPP1 at the newly generated MT ends, whose dynamic instability was concomitantly suppressed. Consistently, MT ends in CSPP1-overexpressing cells were hyper-stabilized, while those in CSPP1-depleted cells were much more dynamic. This CSPP1-elicited stabilization of MTs was demonstrated to be achieved by suppressing intrinsic MT catastrophe and restricting the polymerization. Importantly, CSPP1-bound MTs were resistant to MCAK-mediated depolymerization. These findings delineate a previously uncharacterized CSPP1 activity that integrates MT end capping to orchestrate quiescent MTs.

}, issn = {1759-4685}, doi = {10.1093/jmcb/mjae007}, author = {Wang, Zhikai and Wang, Wenwen and Liu, Shuaiyu and Yang, Fengrui and Liu, Xu and Hua, Shasha and Zhu, Lijuan and Xu, Aoqing and Hill, Donald L and Wang, Dongmei and Jiang, Kai and Lippincott-Schwartz, Jennifer and Liu, Xing and Yao, Xuebiao} } @article {69223, title = {Fluorescence complementation-based FRET imaging reveals centromere assembly dynamics.}, journal = {Molecular Biology of the Cell}, year = {2024}, month = {2024 Feb 21}, pages = {mbcE23090379}, abstract = {

Visualization of specific molecules and their assembly in real time and space is essential to delineate how cellular dynamics and signaling circuit are orchestrated during cell division cycle. Our recent studies reveal structural insights into human centromere-kinetochore core CCAN complex. Here we introduce a method for optically imaging trimeric and tetrameric protein interactions at nanometer spatial resolution in live cells using fluorescence complementation-based F{\"o}rster resonance energy transfer (FC-FRET). Complementary fluorescent protein molecules were first used to visualize dimerization followed by FRET measurements. Using FC- FRET, we visualized centromere CENP-SXTW tetramer assembly dynamics in live cells, and dimeric interactions between CENP-TW dimer and kinetochore protein Spc24/25 dimer in dividing cells. We further delineated the interactions of monomeric CENP-T with Spc24/25 dimer in dividing cells. Surprisingly, our analyses revealed critical role of CDK1 kinase activity in the initial recruitment of Spc24/25 by CENP-T. However, interactions between CENP-T and Spc24/25 during chromosome segregation is independent of CDK1. Thus, FC-FRET provides a unique approach to delineate spatiotemporal dynamics of trimerized and tetramerized proteins at nanometer scale and establishes a platform to report the precise regulation of multimeric protein interactions in space and time in live cells.

}, issn = {1939-4586}, doi = {10.1091/mbc.E23-09-0379}, author = {Dou, Zhen and Liu, Ran and Gui, Ping and Fu, Chuanhai and Lippincott-Schwartz, Jennifer and Yao, Xuebiao and Liu, Xing} } @article {64987, title = {NDP52 tunes cortical actin interaction with astral microtubules for accurate spindle orientation.}, journal = {Cell Research}, volume = {29}, year = {2019}, month = {2019 Jun 14}, pages = {666-79}, abstract = {

Oriented cell divisions are controlled by a conserved molecular cascade involving Gαi, LGN, and NuMA. Here, we show that NDP52 regulates spindle orientation via remodeling the polar cortical actin cytoskeleton. siRNA-mediated NDP52 suppression surprisingly revealed a ring-like compact subcortical F-actin architecture surrounding the spindle in prophase/prometaphase cells, which resulted in severe defects of astral microtubule growth and an aberrant spindle orientation. Remarkably, NDP52 recruited the actin assembly factor N-WASP and regulated the dynamics of the subcortical F-actin ring in mitotic cells. Mechanistically, NDP52 was found to bind to phosphatidic acid-containing vesicles, which absorbed cytoplasmic N-WASP to regulate local filamentous actin growth at the polar cortex. Our TIRFM analyses revealed that NDP52-containing vesicles anchored N-WASP and shortened the length of actin filaments in vitro. Based on these results we propose that NDP52-containing vesicles regulate cortical actin dynamics through N-WASP to accomplish a spatiotemporal regulation between astral microtubules and the actin network for proper spindle orientation and precise chromosome segregation. In this way, intracellular vesicles cooperate with microtubules and actin filaments to regulate proper mitotic progression. Since NDP52 is absent from yeast, we reason that metazoans have evolved an elaborate spindle positioning machinery to ensure accurate chromosome segregation in mitosis.

}, issn = {1748-7838}, doi = {10.1038/s41422-019-0189-9}, author = {Yu, Huijuan and Yang, Fengrui and Dong, Peng and Liao, Shanhui and Liu, Wei R and Zhao, Gangyin and Qin, Bo and Dou, Zhen and Liu, Zhe and Liu, Wei and Zang, Jianye and Lippincott-Schwartz, Jennifer and Liu, Xing and Yao, Xuebiao} } @article {47623, title = {Superresolution imaging reveals structural features of EB1 in microtubule plus-end tracking.}, journal = {Molecular biology of the cell}, volume = {25}, year = {2014}, month = {2014 Dec 15}, pages = {4166-73}, abstract = {

Visualization of specific molecules and their interactions in real time and space is essential to delineate how cellular dynamics and the signaling circuit are orchestrated. Spatial regulation of conformational dynamics and structural plasticity of protein interactions is required to rewire signaling circuitry in response to extracellular cues. We introduce a method for optically imaging intracellular protein interactions at nanometer spatial resolution in live cells, using photoactivatable complementary fluorescent (PACF) proteins. Subsets of complementary fluorescent protein molecules were activated, localized, and then bleached; this was followed by the assembly of superresolution images from aggregate position of sum interactive molecules. Using PACF, we obtained precise localization of dynamic microtubule plus-end hub protein EB1 dimers and their distinct distributions at the leading edges and in the cell bodies of migrating cells. We further delineated the structure-function relationship of EB1 by generating EB1-PACF dimers (EB1(wt):EB1(wt), EB1(wt):EB1(mt), and EB1(mt):EB1(mt)) and imaging their precise localizations in culture cells. Surprisingly, our analyses revealed critical role of a previously uncharacterized EB1 linker region in tracking microtubule plus ends in live cells. Thus PACF provides a unique approach to delineating spatial dynamics of homo- or heterodimerized proteins at the nanometer scale and establishes a platform to report the precise regulation of protein interactions in space and time in live cells.

}, keywords = {Amino Acid Sequence, Cell Movement, Hela Cells, Humans, MCF-7 Cells, Microscopy, Fluorescence, Microtubule-Associated Proteins, Microtubules, Molecular Sequence Data, Protein Transport}, issn = {1939-4586}, doi = {10.1091/mbc.E14-06-1133}, author = {Xia, Peng and Liu, Xing and Wu, Bing and Zhang, Shuyuan and Song, Xiaoyu and Yao, Phil Y and Lippincott-Schwartz, Jennifer and Yao, Xuebiao} }