Monday, 2 April 2012

Cortical dynein and RanGTP in Mitotic Spindle Orientation

by Alan Zhang, s4235621

During mitosis, the mitotic spindle is an important framework of microtubules which align the chromosomes of the cell to maintain even DNA segregation during telophase [1]. Early in mitosis, prophase, spindle poles protrude the early microtubule framework of the mitotic spindle[1]. Researchers of the Whitehead Institute, Kiyomitsu and Cheeseman, have recently published their journal, finding two novel signal gradients that regulate spindle orientation during mitosis of human cells [3]. Previous studies have suggested that cortical dynein, a motor protein, and other regulatory cues, as essential components in mitotic spindle formation, acting on astral microtubules of spindle poles [2][7][9].

With fluorescent microscopy and time-lapse movies the authors have observed dynamic asymmetric localization of cortical dynein-dynactin complexes during spindle orientation [3]. Dynactin is a receptor and activator of dynein which targets it to LNG (leucine glycine asparagine protein)-NuMA (nuclear mitotic apparatus protein) of the cell cortex [3]. Kiyomitsu and Cheeseman found that cortical dynein exhibit forces which pull astral microtubules towards it [3]. The authors discovered that spindle poles emit short ranged inhibitory signals that negatively regulate cortical dynein-dynactin complexes, known as polo-like kinase 1 (plk1) [3]. Consequently, the mitotic spindle undergoes oscillations towards the cell plate, until there is a balance of force [3]. Artificially inhibiting cortical-dynein-dynactin complexes and LGN showed to disrupt the mitotic spindle [3][9][7].
 Figure 1: Time-lapse movies of mitotic spindle during mitosis. Control exhibits normal spindle orientation. LGN RNAi results in the inhibition of LGN at the cell cortex, disrupting spindle orientation. Nocodazole disrupts spindle orientation.

Kiyomitsu and Cheeseman have found another important regulatory signal gradient involving small GTPase Ran (ras-related nuclear protein)  at the chromosome [3]. A RanGEF (guanine-nucleotide exchange factor), called RCC1, binds to the chromosomes which locally enhances formation of RanGTP (the active form of Ran) [3]. The Ran gradient was shown to negatively localise LGN-NuMA of the cell cortex, subsequently dynein-dynactin as well, in the vicinity of chromosomes [3]. This dynamic stabilizes the mitotic spindle lateral axis, as forces from the top and bottom are inhibited. The authors have concluded that RanGTP possibly regulates the ability of LGN-NuMA to interact with the cell cortex [1]. The Ran gradient is known to be involved in a wide range of cellular processes during the cell cycle, such as nuclear transport during interphase and microtubule formation near chromosomes during mitosis [3][4].

Figure 2: Illustration showing the two signal gradients involved during mitotic spindle orientation, Plk1 (red) and RanGTP ( blue) (Kiyomitsu and Cheeseman, 2012).

The journal shows that mitotic spindle orientation is powered by cortical dynein and regulated by two signal gradients during mitosis, Plk1 and the RanGTP [3]. However, many things still remain elusive such as the molecular level of how RanGTP interacts with LNG-NuMA and causes it to delocalise off the cell cortex. Understanding still remains underdeveloped, especially of other eukaryotic cells, such as plants, which don’t have centrosomes [1]. Greater understanding in these processes and other complementary signals can be applied into targeted therapy and chemotherapy for various diseases [6]. Inhibitors and targeting drugs can provide effective therapeutic applications to cancers, which can target specific cells, important signals (such as Plk1, cortical dynein and LGN-NuMA) and microtubules in different cell cycles of cancerous cells to cause mitotic arrest/death [6]. One of the main goals of anti-mitotics (a branch of chemotherapy drugs) is to disrupt the mitotic spindle. Two common classes are taxanes, which promote excessive microtubule growth, and vinca alkaloids, which inhibit mirotubule polymerisation, both leading to cell death. The journal adds input into the dynamics of mitotic spindle orientation and regulation. The authors’ findings have changed the views on mitotic spindle orientation, and have informed the important fundamentals of it, contributing greatly to understanding.

Reference List:
[1] Campbell, NA, Reece, JB & Meyers, N, Biology 8th edition Australian Version, Pearson Education, Australia.
[2] Gusnowski, EM & Srayko, M, 2011, Visualization of dynein-dependent microtubule gliding at the cell cortex: implications for spindle positioning, JCB, no. 3, vol.194, pp. 377-386
[3] Kiyomitsu, T & Cheeseman, IM, 2012, Chromosome- and spindle-pole-derived signals generate an intrinsic code for spindle position and orientation, Nature Cell Biology, No. 13, pp. 311-317
[4] Lonhienne, TG, Forwood, JK, Marfori, M, Robin, G, Kobe, B & Carroll, BJ, 2009, Importin-β Is a GDP-to-GTP Exchange Factor of Ran IMPLICATIONS FOR THE MECHANISM OF NUCLEAR IMPORT, The Journal of Biological Chemistry, No. 284, pp. 22549-22558
[5] Markus, SM & Lee, W, 2011, Microtubule-dependent path to the cell cortex for cytoplasmic dynein in mitotic spindle orientation, BioArchitecture, 1:5, pp 1-7, viewed 2012, 10 March 2012,
[6] Miyamoto, DT, Perlman, ZE, Mitchison, TJ & Shirasu-Hiza, M, 2003, Dynamics of mitotic spindle – potential therapeutic targets, Progress in Cell Cycle Research, vol. 5, pp. 349-360
[7] O'Connell, CB & Wang, Y, 2000, Mammalian Spindle Orientation and Position Respond to Changes in Cell Shape in a Dynein-dependent Fashion, Molecular Biology of the Cell, Vol. 11, pp. 1765-1774
[8] Steuer, ER, Wordeman, L, Schroer, TA & Sheetz, MP, 1990, Localization of Cytoplasmic Dynein to Mitotic Spindles and Kinetochores, Nature, vol. 345, pp. 266 – 268
[9] Vaisberg, EA, Koonce, MP & McIntosh, JR, 1993, Cytoplasmic Dynein Plays a Role in Mammalian Mitotic Spindle Formation, The Journal of Cell Biology, No. 4, Vol. 123, pp. 849-858

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