The function and regulation of microtubule networks
We investigate the function and regulation of microtubule networks in mammalian cells. Microtubules play important roles in a variety of cellular processes, including cell division, cell migration, and neuronal morphogenesis. The microtubule polymer is highly dynamic within cells, and a large number of microtubule-associated proteins (MAPs) interact with microtubules and modulate their dynamics, nucleation, and stability, as well as their interactions with other proteins and organelles. The precise regulation of microtubule dynamics and interactions differs over cell development, the cell cycle, and intracellular space, and is essential to the cellular processes in which microtubules function. Misregulation of these properties can lead to disease progression, and it is thus important to understand how changes in microtubule dynamics facilitate function, and how these changes are regulated.
We primarily investigate these problems in human cancer and non-transformed cell lines, as well as mouse embryonic stem cells and neuronal cells, using a combination of genome modification strategies, live-cell fluorescence microscopy, and quantitative interaction proteomics.
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Areas of current focus are overlapping, but can generally be divided into the following topics:
Mechansims of mitotic spindle assembly and chromosome segregation
When a cell divides, it must segregate all of its chromosomes equally into two daughter cells, and concomitantly segregate cytoplasmic cell fate determinants through cytokinesis. Both chromosome segregation and cytokinesis are coordinated by the mitotic spindle, a microtubule network constructed and disassembled at every cell division. A large number of proteins modulate microtubule nucleation, dynamics, and interactions in order to assemble and position the spindle, align and segregate chromosomes, and coordinate interactions between the ends of microtubules and the cell cortex. For most cells, even the slightest uncorrected error in spindle function can lead to defective segregation of chromosomes or cell fate determinants, often leading to eventual cell death, or worse, activation of oncogenes or loss of tumor suppressors, which may facilitate the path towards cancer. A major focus of the lab is in uncovering the mechanisms and regulatory pathways that guide the formation and function of the mitotic spindle to ensure genome stability.
Bird and Hyman, JCB, 2008
Aguirre-Portolés et al., Cancer Res, 2012
Hubner, Bird et al., JCB, 2010
Microtubules in the regulation of cell migration
The regulation of cell migration is a highly complex process that is often compromised when cancer cells become metastatic. The microtubule cytoskeleton is an important component within several cellular pathways that control cell migration, but how microtubules and microtubule-associated proteins regulate these pathways remains unclear. We have recently discovered a new microtubule-associated protein, GTSE1, that tracks growing microtubule plus-ends in cells and is important for cell migration. We are currently investigating the molecular mechanisms by which microtubules regulate cell migration capacity by studying GTSE1.
Scolz, Widlund et al., PLoS One, 2012
Genome editing and bacterial artificial chromosome (bac) transgenesis techniques
In order to accurately study protein function in living cells, we want to ensure that the regulation and expression of genes we modify are artificially perturbed as little as possible. To this end, we are employing both Cas9/CRISPR-based genome modification techniques and BACs (large genomic DNA constructs) as transgenes. With the Cas9 nuclease we can target specific protein tags or mutations into the genome of the cells being studied. Similarly, BAC transgenes usually deliver physiological expression at endogenous levels because they retain all cis-regulatory elements in the native configuration, and also allow for alternative splice isoforms, translational and miRNA controls, and alternative polyadenylation sites. Both of these methods provide a more accurate way of probing protein function than traditional cDNA-based studies, which often employ viral promotors that are accompanied by problems with deregulation and over-expression.
We have developed new recombineering procedures to modify BACs, in order to tag them with fluorescent markers, introduce targeted point mutations, or modify them to be RNAi-resistant. This allows us to rapidly construct wild-type and mutated RNAi-resistant transgenes, whose gene products we can follow in living mammalian cells under endogenous regulation as the only version expressed in the cell.
We have also collaborated to develop immunoprecipitation-mass spectrometry protocols incorporating the advantages of BAC transgenes to quantitatively identify differences in protein interaction partners and complex formation in cells containing wild-type versus mutant proteins.
Ongoing work in the lab continues to modify and integrate both Cas9- and BAC-based techniques to better assay protein function in live cells.
Bird et al., Nat Methods, 2012
Poser, Sarov et al., Nat Methods, 2008
Hubner, Bird et al., JCB, 2010