George S. Bloom - US grants

The long range goals of the project are to identify in a comprehensive manner the microtubule-associated proteins (MAPs) of the mammalian mitotic spindle, and to determine the functions of these proteins in both the mitotic and interphase stages of the cell cycle. The principal immediate goals will be to identify new spindle MAPs and to evaluate their roles in mitosis. Cultured mammalian cells wil be the main experimental subjects for this work. Microtubules and MAPs will be purified with the aid of taxol from CHO cells synchronized in mitosis and PtK1 cultures enriched in mitotic cells. Monoclonal antibodies will then be made using putative MAPs as antigens. Initially, the antibodies will be used to establish whether the immunoreactive proteins are genuine MAPs by two complementary techniques, immunoblotting and immunofluorescence microscopy. The former technique will be used to follow the fate of immunoreactive proteins through the stages of the taxol-based purification procedure for microtubules and MAPs. Immunofluorescence microscopy will be used to determine the intracellular distributions of these proteins. Immunoreactive proteins that co-purify with microtubules and MAPs, and are shown by immunofluorescence microscopy to be localized on microtubules in cells will be classified as MAPs. Intracellular functions of these proteins, as well as others that may partially fulfill these criteria, will then be probed by antibody microinjection experiments. The object of these studies will be to interfere with specific aspects of mitosis by microinjection of dividing cells with antibodies to particular MAP species, in order to shed light on MAP functions during mitosis. If time permits, non-dividing cells will be microinjected with antibodies to spindle MAPs that are also found on interphase microtubules. This will permit interphase functions of these MAPs to be evaluated. The final set of experiments proposed here will be the screening of various rat tissues by immunoblotting and immunofluorescence microscopy to determine the cellular distributions of MAPs in vivo. These experiments will identify convenient tissue sources for the future purification of individual MAP species, so that biochemical properties of these proteins may be studied in greater detail in subsequent stages of this project. Because microtubules are essential for mitosis, the studies proposed here should further our understanding of how this process occurs during normal development and in malignant cells.

Neurons and neuroendocrine cells possess an intricate cytoplasmic machinery for the transport and release of membrane-bounded organelles. This organelle trafficking serves a multitude of purposes: to supply proteins to the axon, which cannot synthesize its own resident proteins; to deliver secretory products, such as neurotransmitters and catecholamines, to the cell surface; and for retrograde transport of endocytosed trophic factors. Collectively, these types of organelle movements are essential for the development and maintenance of the neuronal phenotype, and for the specialized secretory functions of neuroendocrine cells. Two types of cytoskeletal polymers play pivotal roles in organelle trafficking. Microbutules serve as the tracks for long range organelle motility, while dense meshworks of microfilaments must be traversed by motile organelles near the cell surface and in cytoplasmic regions immediately surrounding microtubules. The broad purpose of the experiments outlined in this proposal is to characterize mechanisms for regulating organelle transport in neuronal and neuroendocrine cells. It is evident that regulation must occur because organelles capable of being transported along microbutules are not always in transit, and often are capable of moving in either direction relative to microtubule polarity. How, then, does an organelle know when to move along a microtubule and which way to travel? Moreover, do changes in the number and organization of microfilaments affect the ability of organelles to travel through the cell, as evidence suggests, or do microfilaments serve different roles in organelle trafficking? Questions such as these will be addressed in the proposed study. Research will focus on three distinct and complementary levels at which regulation is likely to occur. The first Specific Aim concerns the role of phosphorylation in regulating the functions of kinesin. This microtubule- stimulated ATPase is evidently responsible for anterograde transport of membranous organelles in the axon, and for related organelle motility elsewhere in neurons and in other cell types. Recent evidence from the laboratories of the PI and Co-PI has indicated that the kinesin heavy and light chains become phosphorylated in vivo. The effects of phosphorylation on the ATPase, microtubule binding and organelle binding activities of kinesin will be investigated. The second Specific Aim is focused on the role of MAPs in regulating access of kinesin to the microtubule surface, and on the differential abilities of various brain tubulin isoforms to interact with kinesin. The final Specific Aim will deal with a small G protein recently discovered by the PI's laboratory to interact with the cytoskeleton. This G protein will be cloned and sequenced, and its biological role will be characterized by a combination of cell biological and biochemical approaches.

DESCRIPTION: The major hypothesis of this proposal is that folate receptors are expressed on the placenta and neural tube and that folate uptake to the developing fetus is regulated by potocytosis, a process thought to involve glycosol-phosphatidylinositol (GPI)-anchored proteins and caveolae. In addition renal reabsorption of folate from the glomerular filtrate is dependent upon the same process. The specific aims of this study are: (1) to develop a series of autofluorescent folate receptors with differing anchoring schemes or targeting sequences to direct them to caveolae and coated pits. These chimeric receptors will be used to precisely correlate pathway-specific folate biology with what may be artifact-free visualization of folate receptors in live cells; and (2) to use conditional lethal mutant cell lines with inducible defects in coated pit endocytosis to determine how specific molecules accomplish compartmentation and regulation of folate receptors in caveolae rather than coated pits. The experimental design employs cDNA constructs that when transfected into CHO cells produce chimeric proteins containing anchoring domains or targeting sequences, an autofluorescent reporter protein and the alpha-folate receptor. In addition temperature-sensitive mutant cell lines that are defective in coated-pit endocytosis at non-permissive temperatures will be used to study chimeric protein segregation and function. This highly innovative approach to the study of folate receptor biology is likely to provide important new information on receptor localization, recycling and functional mechanism.

DESCRIPTION: (Applicant's Abstract) The neuron has no rival as a cell type that specializes in transport of membrane-bounded organelles (MBOs) along microtubles (MTs). This distinctive trait of the neuron is dictated by its unique morphological and functional properties. During neurogenesis, for example, the formation of dendrites and axons requires sustained delivery of nascent plasma membrane proteins and lipids into growing neurites. These membrane precursors are packages in vesicle whose surface-associated motor molecules move the vesicle along MTs to the distal tips of neurites, where fusion of the vesicles with the plasma membrane promotes neurite elongation. In differentiated neurons, the maintenance of axons is equally reliant upon MTs. Because the axon lacks protein synthesis machinery, but can be more than 10,000 times as long as the diameter of the perikaryon from which it emanates, the axon must constantly be supplied with new resident proteins to replace those that age and degrade. New axonal proteins are synthesized in the perikaryon, and many, like those destined for incorporation into the axolemma, are packaged in vesicles that use motor proteins to move great distances along MTs toward the axon terminal. Likewise, the precursors of synaptic vesicles (SVs), the ultimate specialized products of neurons, are delivered from the cell body, where they are manufactured, to the distal end of the axon by MT- based transport. Finally, endosomal and pre-lysosomal vesicles carry degraded axonal components and edocytosed neurotrophic factors along axonal MTs in the retrograde direction, toward the perikaryon. This renewal application comprises 3 Specific Aims based on progress made during the present funding period. 1) Reconstituted motility systems will be sued to identify and characterize regulatory factors for MBO transport in brian and adrenal chromaffin cells, with emphasis on transport of SVs and chromaffin granules. The hypothesis that MBO transport along MTs is regulated in an organelle-specific manner will be tested. 2) The structure of a newly discovered complex of MTs and protein phosphatase 2A (PP2A) will be determined. A hypothesis to be tested is that MT-bound PP2A regulates MBO transport along MTs by controlling the phosphorylation state of components of the transport machinery or by regulating MT stability. 3) The role of MTs in maintaining the structure of caveolae will be tested. New data indicate that caveolin, a resident protein of caveolae, cycles constitutively between the plasma membrane and the Golgi by a mechanism that requires MTs for transport toward, but not away from the Golgi. A test will be made of the hypothesis that other resident caveolar proteins also normally more between the plasma membrane and the golgi by a MT- dependent cycle. The hypothesis that dynein is the motor for transport of caveolin to the golgi will be also tested, and the mechanism for caveolin transport from the Golgi back to the plasma membrane will be determined.

DESCRIPTION (provided by applicant): The overall goal of this application is to determine how IQGAP1 and other mammalian IQGAPs regulate actin filament nucleation by the Arp2/3 complex, and by extension, cellular motility. In multicellular animals like humans, cell movements and shape changes underly biological process as diverse as organismal development, wound healing, tumor metastasis, and synaptic plasticity. Unraveling the molecular mechanisms that allow cells to move and change shape has therefore been a major goal. It now seems clear that many forms of animal cell motility and morphogenesis are caused by the polarized assembly of branched actin filaments nucleated by the Arp2/3 complex. This activity of Arp2/3 complex is not consitutive. Instead, it relies on activating factors, like N-WASP and other members of the WASP/WAVE family. Likewise, maximal stimulation of Arp2/3 complex by N-WASP requires additional factors that release N-WASP from an autoinhibited state. A few such factors have been discovered within the past few years, most notably activated Cdc42 and PIP2. Recently, the applicant's laboratory discovered that IQGAP1 can potently stimulate actin assembly in vitro by mechanisms that require both N-WASP and Arp2/3 complex. These seminal observations suggest that IQGAP1 is a major, previously unrecognized regulator of the Arp2/3 complex in vivo, and serves as the foundation of the present application. Three Specific Aims are proposed. 1) The molecular mechanisms by which IQGAP1 regulates actin assembly through Arp2/3 complex will be determined by in vitro reconstitution. 2) The hypothesis that mammalian IQGAPs other than IQGAP1 also regulate Arp2/3 complex will be tested. These include IQGAP2, which is known to exist at the protein level, and IQGAP3, which has been inferred from genomic DNA and expressed sequence tags (ESTs). Evidence for expression of IQGAP3 at the protein level will be sought using immunological methods, mass spectrometry, in situ hybridization, and RTPCR. 3) A test will be made of the hypothesis that stimulation of actin assembly by IQGAP1 in cells is triggered by binding of a targetting domain on IQGAP1 to the cytoplasmic tails of ligand-activated cell surface receptors. The regulation of actin assembly by IQGAP1, and the consequences for cell motility will be studied in cultured mammalian cells by light microscopic methods and immunoprecipitation of cellular protein complexes. Similar studies will be initiated for other mammalian IQGAPs shown by pursuit of Specific Aim 2 to regulate actin assembly through the Arp2/3 complex.

? DESCRIPTION (provided by applicant): The most conspicuous molecular and histological landmarks of Alzheimer's disease (AD), excess brain levels of amyloid-? (A?) peptides and abnormally phosphorylated tau, and their respective accumulation into plaques and tangles, have been known for many years. Because mutations that increase total A? or specific, excessively toxic A? variants can cause AD, nearly all efforts to develop effective, disease modifying drugs for AD have been directed at reducing the A? burden in brain. Unfortunately, no such efforts have yet yielded compounds that are sufficiently safe and efficacious to gain FDA approval. To get past this seemingly unscalable wall that has thwarted all efforts to conquer AD so far, new approaches are needed. One such approach is to unravel the seminal signaling pathways that convert normal healthy neurons into neurons that will die before the AD patients themselves. To that end, we recently made major strides towards understanding what may be the most common pathway for neuron death in AD: ectopic cell cycle re-entry (CCR). Whereas fully differentiated normal neurons have permanently exited the cell cycle, up to 5-10% of the neurons in brain regions affected by AD show signs of CCR. These neurons never divide, but instead eventually die and may account for up to 90% of the neuron loss in AD. Building on a prior report that A? oligomers (A?Os) can induce this ectopic CCR, we found that the mechanism is tau-dependent, and entails activation of 4 protein kinases, fyn, mTOR, PKA and CaMKII, which must respectively lead to tau phosphorylation at Y18, S262, S409 and S416. All of this occurs less than a day after exposure of neurons to A?Os. These activated kinases and phospho-tau epitopes represent potential very early biomarkers for AD, and the kinases also represent potential new therapeutic targets. Although we have now defined many steps of the CCR signaling network, a key issue that remains unresolved is why tau phosphorylation at multiple specific sites is necessary to drive post-mitotic neurons back into the cell cycle. Based on this background we propose to use primary mouse neurons, and brain tissue derived from transgenic mice and humans to address 2 interrelated questions. 1) How does tau phosphorylation alter tau structure in a way that enables CCR? 2) By what mechanism does appropriately phosphorylated tau cause CCR? Although the experiments proposed here have been designed in the context of A?O- induced CCR, which is probably unique to AD, tau pathology and neuronal CCR are commonly observed in many other prominent neurodegenerative disorders, such as Parkinson's disease, Huntington's disease and frontotemporal dementias. The successful completion of the project proposed here may therefore be relevant at the basic science level and clinically not only to AD, but to several non-Alzheimer's tauopathies as well.