Browsing by Subject "Microtubules"

Now showing 1 - 9 of 9

Dissecting the Mitotic Golgi Membranes Mediated Microtubule Polymerization
(2019-11-25) Guo, Haijing; Henne, W. Mike; Goodman, Joel M.; Yu, Hongtao; Seemann, Joachim
A properly assembled astral microtubule network is required for correct mitotic spindle orientation, which is important in multiple development processes as it determines cell fate and function. The initiation and growth of astral microtubules was previously attributed to centrosomes and microtubule stabilizing proteins. Here in my dissertation research, I demonstrate that microtubules initiated by mitotic Golgi membranes contribute to the growth of astral microtubules and the proper orientation of the spindle. In turn, the microtubule initiation activity of mitotic Golgi membranes facilitates the proper inheritance of the single copy Golgi apparatus, which is essential in polarized cellular functions, including directional cell migration and secretion. Microtubule assembly is initiated by the Golgi resident protein GM130, which locally activates the spindle assembly factor TPX2 at the mitotic Golgi membranes. GM130 relieves TPX2 from inhibition by competing for importin α binding. The mitotic phosphorylation of importin α on Serine 62 by Cdk1 switches its substrate preference towards GM130 and enables the competition-based activation. The importin α S62A mutant impedes the local TPX2 activation and compromises the astral microtubules, which ultimately leads to misoriented spindles. Blocking of the GM130-importin α-TPX2 activation pathway reduces the astral microtubule growth rate. I also identified that the human GM130 homolog GLP harbors a domain that is highly similar to the TPX2 activating domain of GM130, which could potentially initiate microtubule assembly. My research reveals the novel role of mitotic Golgi membranes in astral spindle organization and the underlying mechanism that regulates this process in a spatio-temporal manner.
Insight into Microtubule Catastrophe and Growth
(2020-08-01T05:00:00.000Z) Kim, Tae Hyungmin; Lin, Milo; Jaqaman, Khuloud; Rice, Luke M.; Yu, Hongtao
Microtubules are hollow cylindrical polymers of αβ-tubulin that have essential roles segregating chromosomes during cell division, organizing the cytoplasm, establishing cellular polarity, and more. These fundamental activities depend critically on dynamic instability, the stochastic switching of microtubules between phases of growth and rapid shrinking. Dynamic instability is itself a consequence of αβ-tubulin GTPase activity and how it affects interactions between αβ-tubulin in the lattice and at the microtubule end. Although predictive molecular understanding of catastrophe remains elusive, the broad outlines of an understanding have been established. Unpolymerized, GTP-bound αβ-tubulin subunits readily associate at the growing tips of the MTs. Once they are incorporated into the lattice, αβ-tubulin GTPase activity is accelerated. The assembly dependence of GTPase activity results in a "stabilizing cap" of GTP-bound αβ-tubulin near the ends of the growing microtubules. Loss of this stabilizing cap triggers catastrophe, the switch from growth to rapid shrinking, because it exposes the more labile GDP-bound microtubule lattice. And, regaining this GTP cap results in microtubule rescue, the switch from shrinking to growing phase. In Chapter 1, I discuss literature background for microtubule dynamics. In Chapter 2, I present a study in which I explored the molecular mechanism behind microtubule catastrophe using computer simulations. By incorporating various candidates for "missing biochemistry" into the computational models, I discovered that lateral long-range interdimer interactions are crucial in correctly predicting catastrophe frequency as a function of the soluble tubulin concentration. In Chapter 3, I discuss a study that directly measures the αβ-tubulin association and dissociation at the microtubule end. We found that the kinetic on-rate of tubulin dimer to a growing microtubule end may be much smaller than previously thought. Next, in chapter 4, I present studies that explore various factors that affect microtubule shrinking and rescue. Finally, in chapter 5, I will summarize my work on these projects and discuss future directions and preliminary results looking to better understand microtubule dynamics.
Investigation of Cell Morphology and Cell-Induced 3-D Matrix Reorganization
(2008-05-13) Kim, Areum; Petroll, W. Matthew
The overall goal is to develop and apply quantification techniques for assessing the underlying pattern of cytoskeletal organization and cell-matrix mechanical interactions in corneal fibroblasts at the sub-cellular level. To specifically study how Rho and Rac regulate sub-cellular mechanical behavior, cells were plated inside 3-D matrices and incubated with activators and/or inhibitors of Rho and Rac and 3-D optical section images were collected simultaneously. Cell morphology, collagen density and orientation were quantitatively studied. The first important finding is that Rho kinase-dependent contractile force generation leads to co-alignment of cells. This process contributes to global matrix contraction and thus may play a central role in cell transformation and force generation during wound healing. In contrast, activation of Rac using PDGF induces dramatic cell elongation without significant matrix reorganization. PDGF may play a role in cell migration during wound healing process since migration requires protrusion of cell extensions, but not necessarily large contractile force. In order to obtain more detailed understanding of how cells reorganize matrices over time, 4-D imaging techniques were used. Cells were plated inside 3-D matrices and time-lapse DIC and LSCM imaging was performed while disrupting cytoskeletal proteins in the presence or absence of the Rho kinase inhibitor. Addition of nocodazole induced rapid microtubule disruption which resulted in Rho activation and cellular contraction. The matrix was pulled inward by retracting pseudopodial processes, and focal adhesions appeared to mediate this process. When Rho-kinase was inhibited, disruption of microtubules resulted in retraction of dendritic cell processes, and rapid formation and extension of lamellipodial processes at random locations along the cell body, eventually leading to a convoluted, disorganized cell shape. These data suggest that microtubules modulate both cellular contractility and local collagen matrix reorganization via regulation of Rho/Rho kinase activity. In addition, microtubules appear to play a central role in dynamic regulation of cell spreading mechanics, morphology and polarity in 3-D culture. Taken together, these experiments demonstrate that quantitative static and dynamic imaging of cells in 3-D matrices is capable of providing unique insights into the role of specific signaling pathways on the underlying pattern of cytoskeletal organization and cell-matrix mechanical interactions.
An Isolated Clasp TOG Domain Suppresses Microtubule Catastrophe and Promotes Rescue
(2019-04-12) Majumdar, Shreoshi; Zhang, Xuewu; Rice, Luke M.; Yu, Hongtao; Tu, Benjamin
Microtubules are heavily regulated dynamic polymers of αβ-tubulin that are required for proper chromosome segregation and organization of the cytoplasm. Polymerases in the XMAP215 family use arrayed TOG domains to promote faster microtubule elongation. Regulatory factors in the CLASP family that reduce catastrophe and/or increase rescue also contain arrayed TOGs. How CLASP TOGs contribute to activity is poorly understood. Using S. cerevisiae Stu1 as a model CLASP, I report structural, biochemical, and reconstitution studies that clarify functional properties of CLASP TOGs. To begin with, I introduce microtubules, their dynamics and regulatory proteins in Chapter 1. In Chapter 2, I discuss how the two TOGs in Stu1 have very different tubulin-binding properties: TOG2 binds to both unpolymerized and polymerized tubulin, and TOG1 binds very weakly to either. I also explore the structure of TOG2 and how it reveals a CLASP-specific residue that likely dictates distinctive tubulin-binding properties. Next, in Chapter 3, I study how, contrary to the expectation that TOGs must work in arrays, the isolated TOG2 domain strongly suppresses microtubule catastrophe and increases microtubule rescue in vitro. Single point mutations on the tubulin-binding surface of TOG2 ablate its anti-catastrophe and rescue activity in vitro, and Stu1 function in cells. Revealing that an isolated CLASP TOG can regulate polymerization dynamics without being part of an array provides insight into the mechanism of CLASPs and diversifies the understanding of TOG function. Finally, in Chapter 4, I will summarize my work and provide insight into future directions.
Mechanistic Analysis of Microtubule Dynamics and Regulation
(2018-10-15) Geyer, Elisabeth Anne; Jaqaman, Khuloud; Rosen, Michael K.; Yu, Hongtao; Rice, Luke M.
Microtubules are critical components in a cells cytoskeletal network, known to form the mitotic spindle which allows for chromosome segregation and cell division and organizing the cytoplasm of non-dividing cells. The quick reorganization of the cytoskeleton relies heavily on the underlying behavior of microtubules, known as dynamic instability. Dynamic instability, the rapid switch between growing and shrinking states of the microtubule, depends on the functional GTPase behavior of the microtubule polymerizing subunits, αβ-tubulin. Recent studies have noted the presence of multiple conformational states of αβ-tubulin in the microtubule lattice, in addition to major conformational changes that occur in αβ-tubulin within the microtubule as compared to free αβ-tubulin in solution. In Chapter 2, I will discuss a study in which I explored the role of the conformational cycle and its impact on microtubule dynamic instability. By studying a mutation in β-tubulin, T238A, I have shown that nucleotide hydrolysis and conformational changes in the lattice are tightly linked and provide allostery throughout the microtubule. Uncoupling the two cycles disrupts the allostery which greatly impacts the rapid transitions normally seen in dynamic instability that allow for fast and decisive structural rearrangements. In Chapter 3, I will discuss a study that aimed to dissect the molecular mechanisms of the yeast microtubule polymerase, Stu2p. In this project, I developed an all-yeast in vitro reconstitution system using total internal reflection fluorescence microscopy which enabled me to study a variety of Stu2 mutants, in the presence of wild-type and mutant yeast tubulin samples. Here, I discovered how the tubulin conformational state can impact Stu2 function and determined a new property of Stu2 in its ability recognize and bind either the microtubule lattice or free tubulin. From these findings, I have proposed a new alternating engagement mechanism to explain how Stu2 functions processively at the microtubule plus end to increase the growth rates of microtubules. Finally, in Chapter 4 I will summarize my work on both of these projects and discuss both future directions and preliminary results looking to solve the structure of human β:T238A microtubules using cryo-EM.
Recombinant αβ-Tubulin and a Simple Computational Model Shed Light on the Molecular Mechanisms of Microtubule Dynamics
(2015-02-06) Piedra, Felipe-Andrés; Yu, Hongtao; Ranganathan, Rama; Ross, Elliott M.; Rice, Luke M.
Microtubules (MTs) are essential to all eukaryotic organisms. They help segregate chromosomes and organize the cytoplasm. MTs are hollow barrels of the protein αβ-tubulin that exhibit a non-equilibrium behavior called dynamic instability: the stochastic switching of single polymers from a state of gradual growth to one of rapid disassembly. Dynamic instability underlies the MT cytoskeleton's rapid reorganizability and enables its diversity of functions. MTs can be reconstituted from purified αβ-tubulin and have been studied in vitro for over 40 years. Over this time, huge strides have been made in the development of an understanding of dynamic instability. Nevertheless, the mechanistic basis of important phenomena like GTP-dependent assembly and GTP hydrolysis-induced conformational change and catastrophe (the switch from growing to shrinking) remain controversial or unexplained. In Chapter 2, I discuss a study in which we used a computational model to investigate the consequences of a new way of thinking about the effect of nucleotide-state on αβ-tubulin and MT assembly. Our results suggest that GDP exposure on the MT plus-end can frustrate elongation and lead to catastrophe. We therefore predicted that GDP to GTP exchange on the MT plus-end might reduce the frequency of catastrophe. We tested our prediction by analyzing the effects of a mutant αβ-tubulin and a GTP analog designed to increase the rate of terminal nucleotide exchange on MT dynamics in vitro. Our experimental results support the results from our model. Thus, we believe that GDP exposure on the MT plus-end increases the likelihood of catastrophe, and can be countered by GDP to GTP exchange. In Chapter 3, I discuss a comparison of yeast and porcine MT dynamics in vitro. My measurements reveal striking differences between yeast and mammalian MT dynamics, and provide new constraints for models of MT dynamics. I conclude my thesis in Chapter 4 with my view of what my work means, what remains to be done and what paths my work has opened for further exploration.
Regulation of the Cytoskeleton by Kinesins
(2013-11-20) Weil, Lauren Melissa; Albanesi, Joseph P.; Cobb, Melanie H.; Conrad, Nicholas; Scherer, Philipp
Kinesins are motor proteins that associate with microtubules. The position of the motor domain has been linked to kinesin function. While amino-terminal and carboxy-terminal localization of the motor domain is linked to cargo transport, kinesins with the motor domain in the middle (M-kinesins) have a role in microtubule depolymerization. The kinesin-13 family consists of four M-kinesins, KIF2A, KIF2B, KIF2C, and KIF24. These proteins regulate the cytoskeleton through their microtubule depolymerizing activity. All four kinesins have reported functions in mitosis, while little is known about their roles in interphase. KIF2A and KIF2C are upregulated in cancer cells and the increased protein expression influences cell migration and invasiveness. In order to understand how KIF2A and KIF2C influence migration, we analyzed the microtubule and actin cytoskeleton in cells manipulated for kinesin expression. We found that depletion of KIF2A increases the number of focal adhesions and stress fibers and results in defects in cell spreading. KIF2A does not influence the dynamics of focal adhesion assembly or disassembly. In contrast, depletion of KIF2C prevents re-formation of focal adhesions. has little or no effect on the actin cytoskeleton. Here we uncovered a functional divergence in regulation of the cytoskeleton between KIF2A and KIF2C. Furthermore, this is the first time that an M-kinesin, which does not transport cargo, has been shown to influence focal adhesion dynamics.
The Substrate and Regulator of Acetyltransferase San
(2010-05-14) Chu, Chih-Wen; Zou, Hui
Lysine acetylation is one of the most common protein modifications in eukaryotes and plays critical roles in numerous cellular events. San is an acetyltransferase required for proper chromosome segregation in Drosophila and human, but little is known about its substrates or upstream regulators. To identify San substrates, I performed affinity purification and demonstrated that tubulin is a San substrate. Tubulins are the building blocks of microtubules, which display dynamic instability. By regulating the rate of microtubule assembly and disassembly, cells organize the microtubule cytoskeleton to accommodate their specific functions. Posttranslational modifications of tubulin have been implicated in regulating microtubule functions. San acetylates beta-tubulin on lysine-252, which only occurs on free tubulin heterodimers. The acetylation-mimicking mutants are incorporated into the microtubule cytoskeleton in HeLa cells without causing any obvious microtubule defect. However, after cold-induced catastrophe, microtubule regrowth is accelerated in San-siRNA cells while the incorporation of the acetylation-mimicking mutant tubulins is severely impeded. The lysine-252 of beta-tubulin localizes at the intradimer interface and interacts with the phosphate group of the alpha-tubulin-bound GTP. Based on these findings, I propose that San regulates tubulin polymerization by acetylating beta-tubulin lysine-252, which neutralizes the positive charge and makes the tubulin heterodimer adopt a conformation that disfavors tubulin polymerization. In addition, I present evidence indicating that the enzymatic activity of San is inhibited by a protein in an ATP-dependent but hydrolysis-independent manner. Purification and identification of this San inhibitor is still under way, and preliminary data from gel filtration chromatography suggest that the inhibitor may be a multi-subunit protein complex.
A TOG:αβ-Tubulin Complex Structure Reveals Conformation-Based Mechanisms for a Microtubule Polymerase
(2012-12-04) Ayaz, Pelin 1983-; Yu, Hongtao; Albanesi, Joseph P.; Rosen, Michael K.; Rice, Luke M.
Stu2p/XMAP215/Dis1 family proteins are evolutionarily conserved regulatory factors that use alpha/beta-tubulin-interacting TOG (tumor overexpressed gene) domains to catalyze fast microtubule growth. Catalysis requires that these polymerases discriminate between unpolymerized and polymerized forms of alpha/beta-tubulin, but how they do so has remained unclear. In this study, we first introduce the polymerization blocked mutants of alpha/beta-tubulins that we developed as unique tools for biochemical studies of alpha/beta-tubulins to avoid the difficulties that has arisen from the self-assembly tendency of tubulins, then we report the structure of the TOG1 domain from Stu2p bound to the plus end polymerization blocked yeast alpha/beta-tubulin we created to facilitate crystallization. Our structure and further biochemical characterizations of the TOG1:alpha/beta-tubulin complex showed that TOG1 binds alpha/beta-tubulin in a way that excludes equivalent binding of a second TOG domain. Furthermore, TOG1 preferentially binds a “curved” conformation of alpha/beta-tubulin that cannot be incorporated into microtubules, contacting α- and β-tubulin surfaces that do not participate in microtubule assembly. Conformation-selective interactions with alpha/beta-tubulin explain how TOG-containing polymerases discriminate between unpolymerized and polymerized forms of alpha/beta-tubulin, and how they selectively recognize the growing end of the microtubule.