Tim Stearns - US grants

DESCRIPTION (Verbatim from the applicant's abstract): The long term objective of this proposal is to understand the molecular basis of microtubule organizing center function, which is essential to generating a polar microtubule cytoskeleton to carry out the important tasks of chromosome segregation, cell motility, organelle transport, and nuclear migration. We have only just begun to answer some of the central questions in this field: How does the organizing center nucleate the growth of microtubules? How is nucleation temporally and spatially regulated? Which molecules are important for which functions in the organizing center? Cancer cells often have defects in microtubule organizing center size and number, possibly leading to genetic instability, demonstrating the importance of these questions. To further our understanding of the microtubule organizing center (called the centrosome in animal cells) I will focus primarily on the structure and function of gamma-tubulin and the large macromolecular assembly of which it is part, gamma-tubulin complex (also called the gamma-tubulin ring complex, or gamma-TuRC). The gamma-tubulin complex is required for microtubule nucleation, and has been proposed to act as a direct template for the polymerization of microtubules. The gamma-tubulin complex is made up of gamma-tubulin plus five different polypeptides. This remarkable complex must interact both with microtubules and the centrosome, placing it at the center of microtubule organization. We have found that all five of the gamma-tubulin complex proteins, or GCPs, belong to a new protein superfamily. The importance of gamma-tubulin in microtubule organization has led us to search for new tubulins in the mammalian genome and we have discovered two, d(delta)-tubulin and e(epsilon)-tubulin. Epsilon tubulin has not been described before in any organism, and its properties suggest a role in centrosome function as well. We will attack four specific aims in the next project period: 1) Determine the subunit composition and structure of the gamma-tubulin complex. 2) Define the interactions among the gamma-tubulin complex proteins, and their role in the gamma-tubulin complex. 3) Characterize the interaction of the gamma-tubulin complex with the microtubule and the centrosome. 4) Characterize the role of epsilon-tubulin in centrosome function.

DESCRIPTION (provided by applicant): The central problems addressed in the proposed research are how the centrosome is organized, how it duplicates once per cell cycle, and how we go from the parts list for the organelle to a more complete understanding of how it works. The centrosome nucleates microtubules and helps to organize those microtubules to create useful arrays, including the mitotic spindle and the cilium. Work from my lab and others over the last 20 years at Stanford has identified molecules involved in microtubule nucleation, the central regulators of centrosome duplication, important structural proteins involved in duplication, and control mechanisms that govern centrosome number and link the centrosome and the cilium. Interest in the centrosome is strong because of the correlation between centrosome abnormalities and the development of cancer, and because of the role of the centrosome in ciliopathies, diseases related to primary cilium function, and microcephaly, a defect in neuronal development. The proposed experiments make use of the strengths that we have developed in reagents and assays for studying centrosome structure, function and duplication in different experimental systems. We have chosen to focus on the process in animal cells, tissues and extracts as they are the systems most relevant to our desire to understand the human centrosome in normal cell division, and in disease. Three specific aims are addressed in this proposal: Specific Aim 1 - Identify and characterize the interactions that define the centriole origin of duplication Specific Aim 2 - Characterize the mode of action of Mdm1, a unique negative regulator of centriole duplication. Specific Aim 3 - Create centriole-less mammalian cells to address the function of the centrosome in the cell cycle, cell signaling, and nuclear functions.

DESCRIPTION (provided by applicant): The central problems addressed in the proposed research are how the centrosome is assembled, how it duplicates once per cell cycle, and what happens in cells in which centrosome number is aberrant. The centrosome nucleates microtubules and organizes those microtubules to create a useful array. The centrosome is the major microtubule organizing center in animal cells, and is present in a single copy at the beginning of the cell cycle. The centrosome duplicates in S phase, and the two resulting centrosomes help to organize the two poles of the mitotic spindle. Work from my lab and others over the last ten years has identified molecules involved in microtubule nucleation, the central regulators of centrosome duplication, important structural proteins involved in duplication, and control mechanisms that control centrosome number and link the centrosome and the cell cycle. Interest in the centrosome has grown recently because a correlation has been established between centrosome abnormalities and the development of cancer. Cancer cells often have extra centrosomes, which is likely to contribute to the genomic instability and rapid evolution characteristic of this disease. The proposed experiments make use of the strengths that we have developed in reagents and assays for studying centrosome structure, function and duplication. I address four specific aims in this proposal: 1) Characterize the block to centrosome reduplication. We will test models for the mechanism of the block, determine whether cancer cells have defects in the block, and test whether the block can be overcome by manipulation of potential regulators. 2) Investigate the relationship between centrosome number, ploidv, and genome instability. We will use cell fusion methods to create cells of defined centrosome number and ploidy, examine the progression and outcome of mitosis in these cells, and compare normal and cancer cells in their response to centrosome and ploidy abnormalities. 3) Define the molecular interactions of the gamma-tubulin ring complex with microtubules and with the centrosome. We will use purified components to determine the molecular interactions between the gamma-tubulin ring complex, the microtubule, and the centrosome. 4) Investigate the role of delta-tubulin and epsilon-tubulin in centrosome function and duplication. We will characterize the interactions between delta-tubulin, epsilon-tubulin and the other known tubulins, and define the function of delta-tubulin in vitro using assays in frog egg extracts, and in vivo in human cells.

DESCRIPTION (provided by applicant): Mutations in TSC1 and TSC2 cause tuberous sclerosis complex (TSC), a multi-systemic genetic disease characterized by a variety of symptoms such as non-malignant brain or heart tumors, kidney and lung cysts, and neurocognitive defects. TSC1 and TSC2 have well-characterized roles in regulating cellular energy balance and protein translation, but also a number of non-canonical functions, for example in primary cilium formation. However, these functions remain poorly understood and it is currently unknown to what extent each of these TSC1/2 functions contributes to the development of TSC. Previous results from our and other research groups suggest that TSC1 and TSC2 may exert important aspects of their functions at the centrosome and/or in the context of regulating primary cilium structure and function. We hypothesize that TSC is, at least in part, a ciliopathy and that compromising this TSC- centrosome-primary cilium axis may contribute substantially to the development of TSC. Therefore, we aim to characterize the functions of TSC1/2 at the centrosome and to correlate impaired TSC1/2 functions with alterations in ciliary signaling. We will use genome-engineered mouse and human cell lines to test which of the canonical and non- canonical TSC1 or TSC2 functions can be rescued by versions of TSC1/2 that strictly localize to the centrosome in cells that otherwise lack TSC1 or TSC2 function, respectively. To control for TSC1/2 functions that are specifically linked to their centrosomal localization, we will test these functions in cells that lack centrosomes, but express wt TSC1/2. To test whether TSC1/2 loss-of-function phenotypes are a consequence of alterations in centrosome or primary cilium structure or function, we will induce centrosome overduplication and formation of supernumerary primary cilia in cells that have normal TSC1/2 function and test whether these cells mimic TSC1/2-associated phenotypes. In a complementary approach, we will use the BioID proximity labeling method in combination with centrosome purification to identify proteins that are spatially linked to centrosomal TSC1 and TSC2. We will use this approach to create the centrosomal interaction map of TSC1 and TSC2. Ultimately, the proposed work will help in developing diagnostic tools and therapies for TSC that specifically target the TSC1/2 functions that are relevant to the development of TSC phenotypes. In particular, unraveling the functional relationship between mutations in TSC1/2 and disrupted ciliary signaling will open up new routes for TSC treatments that aim to correct these signaling defects, for example by drugs that act as agonists for ciliary signaling receptors. Therefore, harnessing the TSC- centrosome-primary cilium axis for therapeutic purposes may have a significant impact on TSC treatment.

Functional Compartmentalization of Hedgehog Signal Transduction in Primary Cilia Abstract Primary cilia are small, antenna-like organelles critical for vertebrate development and physiology. Defects in cilia result in human diseases called ciliopathies, characterized by a wide spectrum of phenotypes, highlighting their important role in multiple cell types and organs. Although not completely surrounded by a membrane, cilia form a distinct compartment that receives and transmits extracellular signals. Their function critically depends on the dynamic changes in protein composition and localization. The Hedgehog (Hh) signaling transduction, essential for embryonic development, adult tissue regeneration and cancer, largely takes place at primary cilia. The Hh-transduction proteins Smoothened (Smo), Patched1 (Ptch1), Suppressor of Fused (SuFu) and Gli2 shuttle into and out of cilia when cells receive the Hh signal, and changes in localization correlate with pathway activation. The molecular basis for most of these steps is not clear. In this proposal, we will build upon our previous work in Hh signal transduction and in the cell biology of cilia and centrosomes, to answer unresolved questions about protein compartmentalization in cilia and Hh signal transduction. We will apply high-resolution optical microscopy, molecular genetics and novel biochemical methods to determine how specific interactions with ciliary protein complexes control Hh-transducer dynamics in cilia, and, more broadly, how ciliary responsiveness is established; investigate how Ptch1 interactions within the cilium regulate Smo activation; finally, we will elucidate how removal of Hh-transducers from cilia is controlled, extending our recent discovery of Rilp-like proteins as regulators of this process. In summary, we propose to establish a high-resolution, spatially and temporally resolved picture of Hedgehog signaling in the primary cilium. We believe that only by having such an in- depth analysis will it be possible to understand the molecular basis of Hedgehog signaling. !

Abstract The centrosome-cilium complex is a critical organelle of animal cell function. The centrosome is the main microtubule organizing center of animal cells, and the centrioles within the centrosome have a unique structure that allows them to serve as initiators of the assembly of a cilium. Primary cilia are small, antenna-like organelles critical for vertebrate development and physiology. Defects in the centrosome-cilium complex result in human diseases called ciliopathies, characterized by a wide spectrum of phenotypes, highlighting their important role in multiple cell types and organs. This complex forms a distinct compartment of the cell, with a highly polarized microtubule cytoskeleton directing intracellular traffic to and from it, and a specialized segment of the plasma membrane surrounding part of it. In most cells, there is only one centrosome-cilium complex, but this is often altered in cells from a range of diseases, and in cells that have specific developmental programs to amplify centrioles. The function of cilia critically depends on the dynamic changes in protein composition and localization. In particular, Hedgehog (Hh) signaling transduction, essential for embryonic development, adult tissue regeneration and cancer, largely takes place at primary cilia, and involves movements of the signaling proteins into and out of the cilium in response to signal. We have defined six fundamental questions that drive the proposed research. 1) How do the specialized compound microtubules of centriole form, and how do they specifically form only at the centriole? 2) How are centrioles functionalized to carry out the essential roles of cilium formation, centrosome formation, duplication? 3) What are the consequences of failure to maintain centriole number, and what homeostatic mechanisms exist that could facilitate a return to the normal state? 4) What mechanisms promote centriole amplification and loss in specific differentiated contexts? 5) What is the behavior of signaling proteins in the cilium, at the single molecule level, and how does it change in response to activation of the pathway? 6) How are cilia disassembled and is the same mechanism used in different contexts and across evolution? We will use a combination of advanced imaging, cell culture and in vitro differentiation models, combined with sophisticated molecular biology to address these questions. Successful outcomes in these experiments will inform our understanding of centrosome-cilium defects in disease states, including ciliopathies and cancer, and potentially lead to new therapeutic approaches. !

In this grant, we propose to understand the molecular mechanisms of dendrite morphogenesis and function. Dendrite morphogenesis determines the connectivity of neurons. We are using a model cell (PVD in C elegans) to study this question. PVD is a proprioceptive neuron that senses muscle contraction and regulates animal movement. In our previous work, we identified the extracellular ligands and their receptor on PVD that guide the dendrite growth and branching. Here, we propose to understand how the receptor-ligand interaction triggers signaling mechanisms and leads to cytoskeletal modifications which eventually drives the morphogenesis events. We will also study how the neurons regulate receptor signaling using a drug target protein called KPC-1 to control guidance decisions. We will also understand how the PVD neurons sense muscle contraction using a putative mechanosensitive channel and how it regulates neuromuscular activity through a surprising neural circuit feedback mechanism. Through these experiments, we will gain insights in the molecular logic of dendrite development. We will identify novel mechanosensitive channels that are important for body movement regulation.