(A*STAR PROGRAMME) Cell cycle control of dynein-adaptor interactions

University of Manchester Supervisors: Prof Viki Allan, Prof Martin Lowe. A*STAR Supervisor: Brian Burke (IMB).

The microtubule motor, cytoplasmic dynein, drives the movement of a huge range of different cargoes both during interphase and when cells are dividing. During meiosis, dynein-driven movement of chromosomes promotes pairing of homologous chromosomes: without this pairing, meiosis I fails. Dynein is recruited to the cytoplasmic face of the nuclear envelope (NE) at early stages of meiotic prophase I by binding to the outer nuclear envelope protein KASH5. KASH5 also interacts with an inner nuclear envelope protein that in turn binds to the telomeres of meiotic chromosomes, so generating a connection from dynein in the cytoplasm through to the chromosome inside the nucleus. KASH5 is essential for meiosis, and without it, no functional sperm or eggs are produced.

Dynein also plays many important roles during mitosis in all cells. For example, it is recruited to the NE from late G2 on through prophase of mitosis, where it is needed for centrosome separation and facilitates nuclear envelope breakdown. In mitotic cells, dynein is recruited to the NE at late G2 by BicD2 and in mitotic prophase by CENP-F/Nde1/Nde1L. Dynein is also vital for establishing and maintaining the structure of the spindle.

We have generated a HeLa line stably expressing GFP-KASH5 as a model for studying KASH5-dynein interactions. Using interphase cells, we have found that KASH5 is a new adaptor for dynein that promotes the formation of tripartite complexes containing dynein and its regulatory complex dynactin, along with KASH5. KASH5 interacts with the light intermediate chains (LICs) of dynein. The expression of a cytosolic, truncated form of KASH5 (which sequesters dynein/dynactin) strongly inhibits dynein’s interphase roles as a membrane motor, but does not affect spindle morphology, even though this depends on dynein. This striking result suggests that KASH5 no longer binds dynein in pro-metaphase/metaphase. Interestingly, the expression of dominant negative form of another dynein adaptor protein, BicD2, also has no effect on spindle assembly or morphology. We propose that dynein binds one set of adapters in interphase, and another set as cells enter prometaphase.

We will test this hypothesis by determining how dynein’s ability to bind two interphase adaptors—KASH5 and BicD2—and one mitotic adaptor—spindly—is controlled through the cell cycle. We will use biochemical approaches to assess complex formation, and mass spectrometry to identify changes in phosphorylation of dynein LICs and the adaptors. Based on these results, we will make phosphorylation site mutants to test their effects on complex formation.

We will also investigate the effect that recruiting additional dynein to the NE via KASH5 has on prophase functions of dynein, and on cell cycle progression and nuclear envelope reassembly as cells exit mitosis. To do this, we will use live cell imaging of the NE in combination with super-resolution microscopy.