On the role of the phosphatidylinositol transfer protein alpha in the nuclear import of phosphatidylinositol

Lipid metabolism within the nuclear matrix of mammalian cells is robust. Numerous lipid-signaling pathway components are found within purified nuclear fractions, depleted of nuclear envelope and cellular debris using detergents ("endonuclear" fractions). Interestingly, membrane-stripped nuclei also contain measurable quantities of the substrate lipids, notably phospholipids (PLs), despite the absence of detectable bilayer structures. According to published work, these PLs are i) major constituents of the endonuclear space (10-16% by volume), ii) distinct from cytosolic/cellular lipids in molecular species profiles and relative abundance, and iii) generated or consumed in response to physiological cues. The abundance and diversity of PL molecular species in nuclei suggests endonuclear lipids not only signal within the nuclear space, but also impact nuclear structure and function on the whole. That the elements of functional endonuclear phosphoinositide signaling pathways (enzymes and PLs) are found within nuclei has received much attention, as their cytosolic counterparts regulate numerous cellular events. While new evidence suggests that nuclear-generated phosphoinositides participate as cofactors in essential nuclear processes, there are still unanswered questions regarding the regulation, location, and organization of endonuclear phosphoinositides. Phosphatidylinositol, the PL from which all phosphoinositides are generated, enters the nuclear space via an unknown mechanism. This dissertation originated in testing a candidate importer protein, the phosphatidylinositol transfer protein alpha, in the nuclear supply of Phosphatidylinositol. Such experiments require envelope-stripped nuclei that meet high purity standards. Initial attempts at purifying nuclei via published protocols were unsuccessful, and a new method for purifying nuclei from mouse embryonic fibroblasts for use in this context is described. Nuclei purified according to the new protocol meet an expanded, quantitative quality control suite. These highly purified nuclei contain several orders of magnitude less PL than previously reported in LC/MS/MS mass analyses. In addition, pulse-labeling and comparative dynamic lipidomic studies of mouse embryonic fibroblast nuclei, either wild type or genetically knocked-out for Phosphatidylinositol transfer protein alpha, purified with this new protocol demonstrated that our candidate shuttle protein was not an obligate requirement in the import of Phosphatidylinositol. Overall, this dissertation demonstrates that proper execution of nuclear preparations is an essential component of studies of endonuclear lipid regulation

Tektin 2 is required for central spindle microtubule organization and the completion of cytokinesis

During anaphase, the nonkinetochore microtubules in the spindle midzone become compacted into the central spindle, a structure which is required to both initiate and complete cytokinesis. We show that Tektin 2 (Tek2) associates with the spindle poles throughout mitosis, organizes the spindle midzone microtubules during anaphase, and assembles into the midbody matrix surrounding the compacted midzone microtubules during cytokinesis. Tek2 small interfering RNA (siRNA) disrupts central spindle organization and proper localization of MKLP1, PRC1, and Aurora B to the midzone and prevents the formation of a midbody matrix. Video microscopy revealed that loss of Tek2 results in binucleate cell formation by aberrant fusion of daughter cells after cytokinesis. Although a myosin II inhibitor, blebbistatin, prevents actin-myosin contractility, the microtubules of the central spindle are compacted. Strikingly, Tek2 siRNA abolishes this actin-myosin–independent midzone microtubule compaction. Thus, Tek2-dependent organization of the central spindle during anaphase is essential for proper midbody formation and the segregation of daughter cells after cytokinesis

Amphiastral Mitotic Spindle Assembly in Vertebrate Cells Lacking Centrosomes

The role of centrosomes/centrioles during mitotic spindle assembly in vertebrates remains controversial. In cell-free extracts and experimentally derived acentrosomal cells, randomly oriented microtubules (MTs) self-organize around mitotic chromosomes and assemble anastral spindles [1, 2, 3]. However, vertebrate somatic cells normally assemble a connected pair of polarized, astral MT arrays – termed an amphiaster (“a star on both sides” [4]) – that is formed by the splitting and separation of the microtubule-organizing center (MTOC) well before nuclear envelope breakdown (NEB) [5]. Whether amphiaster formation requires splitting of duplicated centrosomes is not known. We found that when centrosomes were removed from living vertebrate cells early in their cell cycle, an acentriolar MTOC re-assembled, and prior to NEB, a functional amphiastral spindle formed. Cytoplasmic dynein, dynactin, and pericentrin are all recruited to the interphase aMTOC, and the activity of kinesin-5 is needed for amphiaster formation. Mitosis proceeded on time and these karyoplasts divided in two. However, ~35% of aMTOCs failed to split/separate before NEB, and these entered mitosis with persistent monastral spindles. The chromatin-mediated RAN-GTP pathway could not restore bipolarity to monastral spindles, and these cells exited mitosis as single daughters. Our data reveal the novel finding that MTOC separation and amphiaster formation does not absolutely require the centrosome, but in its absence, the fidelity of bipolar spindle assembly is highly compromised

Quantitative profiling of the endonuclear glycerophospholipidome of murine embryonic fibroblasts

A reliable method for purifying envelope-stripped nuclei from immortalized murine embryonic fibroblasts (iMEFs) was established. Quantitative profiling of the glycerophospholipids (GPLs) in envelope-free iMEF nuclei yields several conclusions. First, we find the endonuclear glycerophospholipidome differs from that of bulk membranes, and phosphatidylcholine (PtdCho) and phosphatidylethanolamine species are the most abundant endonuclear GPLs by mass. By contrast, phosphatidylinositol (PtdIns) represents a minor species. We also find only a slight enrichment of saturated versus unsaturated GPL species in iMEF endonuclear fractions. Moreover, much lower values for GPL mass were measured in the iMEF nuclear matrix than those reported for envelope-stripped IMF-32 nuclei. The collective results indicate that the nuclear matrix in these cells is a GPL-poor environment where GPL occupies only approximately 0.1% of the total nuclear matrix volume. This value suggests GPL accommodation in this compartment can be satisfied by binding to resident proteins. Finally, we find no significant role for the PtdIns/PtdChotransfer protein, PITP\uf4, in shuttling PtdIns into the iMEF nuclear matrix.-Tribble, E. K., P. T. Ivanova, A. Grabon, J. G. Alb, Jr., I. Faenza, L. Cocco, H. A. Brown, and V. A. Bankaitis. Quantitative profiling of the endonuclear glycerophospholipidome of murine embryonic fibroblasts

Home Owners' Loan Corporation Neighborhood Grades Composite Shapefile, Manhattan, 1937

This polygon shapefile represents neighborhood boundaries and Home Owners' Loan Corporation (HOLC) "grades" and notes for major metropolitan areas in Manhattan in 1937. The layer is a polygon rendering of a print map that has been scanned, vectorized, and transcribed by the University of Richmond Digital Scholarship Lab. Using data and evaluations organized by local real estate professionals, lenders, developers, and real estate appraisers in Manhattan, the HOLC assigned "grades" to residential neighborhoods that reflected their "mortgage security" that would then be visualized on color-coded maps. Neighborhoods receiving the highest grade of "A"--colored green on the maps--were deemed minimal risks for banks and other mortgage lenders when they were determining who should received loans and which areas in the city were safe investments. Those receiving the lowest grade of "D," colored red, were considered "hazardous." The creation and circulation of these maps has been widely proven to contribute to the intergenerational oppression of Black Americans and people of color in Manhattan. Refer to the documentation for geoprocessing lineage and more information and visit https://dsl.richmond.edu/panorama/redlining/#loc=5/39.1/-94.58&text=intro/

Home Owners' Loan Corporation Neighborhood Grades Composite Shapefile, United States, 1935

This polygon shapefile represents neighborhood boundaries and Home Owners' Loan Corporation (HOLC) "grades" and notes for major metropolitan areas in the United States in 1935. The layer is a polygon rendering of many print maps that have been scanned, vectorized, and transcribed by the University of Richmond Digital Scholarship Lab. Using data and evaluations organized by local real estate professionals, lenders, developers, and real estate appraisers in each city in the 1930s, the HOLC assigned "grades" to residential neighborhoods that reflected their "mortgage security" that would then be visualized on color-coded maps. Neighborhoods receiving the highest grade of "A"--colored green on the maps--were deemed minimal risks for banks and other mortgage lenders when they were determining who should received loans and which areas in the city were safe investments. Those receiving the lowest grade of "D," colored red, were considered "hazardous." The creation and circulation of these maps has been widely proven to contribute to the intergenerational oppression of Black Americans and people of color in the United States. Refer to the documentation for geoprocessing lineage and more information and visit https://dsl.richmond.edu/panorama/redlining/#loc=5/39.1/-94.58&text=intro/

Home Owners' Loan Corporation Neighborhood Grades Composite Shapefile, Manhattan, 1937

This polygon shapefile represents neighborhood boundaries and Home Owners' Loan Corporation (HOLC) "grades" and notes for major metropolitan areas in Manhattan in 1937. The layer is a polygon rendering of a print map that has been scanned, vectorized, and transcribed by the University of Richmond Digital Scholarship Lab. Using data and evaluations organized by local real estate professionals, lenders, developers, and real estate appraisers in Manhattan, the HOLC assigned "grades" to residential neighborhoods that reflected their "mortgage security" that would then be visualized on color-coded maps. Neighborhoods receiving the highest grade of "A"--colored green on the maps--were deemed minimal risks for banks and other mortgage lenders when they were determining who should received loans and which areas in the city were safe investments. Those receiving the lowest grade of "D," colored red, were considered "hazardous." The creation and circulation of these maps has been widely proven to contribute to the intergenerational oppression of Black Americans and people of color in Manhattan. Refer to the documentation for geoprocessing lineage and more information and visit https://dsl.richmond.edu/panorama/redlining/#loc=5/39.1/-94.58&text=intro/

Home Owners' Loan Corporation Neighborhood Grades Composite Shapefile, United States, 1935

This polygon shapefile represents neighborhood boundaries and Home Owners' Loan Corporation (HOLC) "grades" and notes for major metropolitan areas in the United States in 1935. The layer is a polygon rendering of many print maps that have been scanned, vectorized, and transcribed by the University of Richmond Digital Scholarship Lab. Using data and evaluations organized by local real estate professionals, lenders, developers, and real estate appraisers in each city in the 1930s, the HOLC assigned "grades" to residential neighborhoods that reflected their "mortgage security" that would then be visualized on color-coded maps. Neighborhoods receiving the highest grade of "A"--colored green on the maps--were deemed minimal risks for banks and other mortgage lenders when they were determining who should received loans and which areas in the city were safe investments. Those receiving the lowest grade of "D," colored red, were considered "hazardous." The creation and circulation of these maps has been widely proven to contribute to the intergenerational oppression of Black Americans and people of color in the United States. Refer to the documentation for geoprocessing lineage and more information and visit https://dsl.richmond.edu/panorama/redlining/#loc=5/39.1/-94.58&text=intro/