The Role of the MRX Complex and the Non-homologous End Joining DNA Repair Pathway in Mitochondrial Genome Stability and Repair

Mitochondria are required for cellular respiration, which is essential in the production of ATP. Mitochondrial genome maintenance is necessary for the continued function of the mitochondrion. Deletions within the mitochondrial DNA (mtDNA) have been shown to be associated with a variety of human neuromuscular and age-related diseases. In this study we investigated the role of the MRX complex and the non-homologous end joining (NHEJ) DNA repair pathway in mitochondrial genome stability and repair. Specifically, we investigated the role of the MRX complex and the NHEJ pathway in the occurrence of spontaneous mitochondrial direct repeat-mediated deletions, nuclear direct repeat-mediated deletions, mitochondrial point mutations, nuclear point mutations, and spontaneous respiration loss using fluctuation analysis in the budding yeast, Saccharomyces cerevisiae. In this study, we have demonstrated that spontaneous mitochondrial direct repeat-mediated deletions are reduced 75 fold (

KIF7 and Microtubule Dynamics Function to Regulate Cellular Proliferation and Cell Cycle Progression

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Biomedical Genetics, 2015.Respiratory diseases such as lung cancer, COPD, and asthma are the second leading cause of death in the United States. These diseases are heterogeneous and arise from genetic factors, environmental hazards, or congenital abnormalities that persist throughout life. An increased understanding of the genes and cellular mechanisms regulating respiratory system homeostasis and regeneration should provide information for the development of future therapeutics. We performed a forward genetic screen in mice aimed at identifying novel genes and mutation required for respiratory system function in the laboratory mouse, Mus Musculus. We indentified a mouse mutant line with tissues patterning, and tissue hyperplasia phenotypes. We determined that the causative mutation occurred within the cilia and microtubule associated gene Kinesin family member 7, Kif7. By characterizing the phenotypes of Kif7 mutant mice and Kif7 depleted cells, we identified several novel functions for this molecule. We show that the gene Kif7 regulates cell proliferation, cellular density, and intracellular signaling within the epithelial and mesenchymal cells of the respiratory airway. We expand on the known role for KIF7 by showing that this protein functions to maintain cytoskeletal microtubule organization and controls fibroblast cellular density and to regulate cell cycle progression and cell signaling in non-ciliated secretory cells. Furthermore, we show that microtubules function to regulate the abundance and activity of several factors known to be required for proper cell cycle timing. We propose that KIF7 and microtubule dynamics hone cellular signaling necessary for control of the balance between cell proliferation and cell cycle exit, and we provide evidence that Kif7 has a critical role in the maintenance of the respiratory system in postnatal life

KIF7 Controls the Proliferation of Cells of the Respiratory Airway through Distinct Microtubule Dependent Mechanisms

<div><p>The cell cycle must be tightly coordinated for proper control of embryonic development and for the long-term maintenance of organs such as the lung. There is emerging evidence that <i>Kinesin family member 7</i> (<i>Kif7</i>) promotes <i>Hedgehog</i> (<i>Hh</i>) signaling during embryonic development, and its misregulation contributes to diseases such as ciliopathies and cancer. <i>Kif7</i> encodes a microtubule interacting protein that controls <i>Hh</i> signaling through regulation of microtubule dynamics within the primary cilium. However, whether <i>Kif7</i> has a function in nonciliated cells remains largely unknown. The role <i>Kif7</i> plays in basic cell biological processes like cell proliferation or cell cycle progression also remains to be elucidated. Here, we show that <i>Kif7</i> is required for coordination of the cell cycle, and inactivation of this gene leads to increased cell proliferation <i>in vivo</i> and <i>in vitro</i>. Immunostaining and transmission electron microscopy experiments show that <i>Kif7</i>^{<i>dda/dda</i>} mutant lungs are hyperproliferative and exhibit reduced alveolar epithelial cell differentiation. KIF7 depleted C3H10T1/2 fibroblasts and <i>Kif7</i>^{<i>dda/dda</i>} mutant mouse embryonic fibroblasts have increased growth rates at high cellular densities, suggesting that <i>Kif7</i> may function as a general regulator of cellular proliferation. We ascertained that in G1, <i>Kif7</i> and microtubule dynamics regulate the expression and activity of several components of the cell cycle machinery known to control entry into S phase. Our data suggest that <i>Kif7</i> may function to regulate the maintenance of the respiratory airway architecture by controlling cellular density, cell proliferation, and cycle exit through its role as a microtubule associated protein.</p></div

KIF7 is Expressed in the fetal and postnatal lung.

<p>(<b>A.-F’.</b>) Confocal immunofluorescent staining of KIF7 in the respiratory epithelium. (<b>A.-B.</b>) KIF7 co-localization with CDH1 (E-cadherin) at embryonic day E14.5. Scale bars are 50 and 30 microns respectively. <b>B</b> is a zoom of the boxed region (<b>C.</b>) KIF7 co-localization with acetylated alpha tubulin (a marker of the primary cilium) at E14.5 within the lung epithelium. Scale bar is 10 microns. <b>C’</b> and <b>C”</b> are zooms from the boxed regions of mesenchymal and epithelial cells in <b>C</b>. (<b>D.-E.</b>) KIF7 co-localization with PDPN (a marker for the type 1 AEC) at postnatal day 0. <b>E</b> is a zoom of the boxed region. Scale bars are 50 and 10 microns respectively. (<b>F-F’</b>.) KIF7 co-localization with FASN (a marker for the type 2 AEC) at postnatal day 7. Scale bar is 5 microns. <b>F’</b> is a zoom of <b>F.</b></p

KIF7 Regulates <i>Hh</i> Signaling, Cell density, and Cell cycle Exit in Fibroblasts Within the Neonatal Lung.

<p>(<b>A.-D.</b>) Tissue sections from control and <i>Kif7</i>^<i>dd/dd</i> mutant embryos were immunostained with the cell proliferation marker Ki67. (<b>A.+B.</b>) Sections from postnatal day 0 (P0) control and <i>Kif7</i>^<i>dd/dd</i> mutant lungs. (<b>C.+D.</b>) Representative tissue sections from E18.5 control and <i>Kif7</i>^<i>dd/dd</i> mutant lungs. Note that P0 <i>Kif7</i>^<i>dd/dd</i> mutant lungs are denser and have more Ki67⁺ cells than E18.5 control lungs. Scale bar is 50 microns. (<b>E.-F.</b>) <i>Gli1-lacZ Hh</i> reporter mouse line was crossed into <i>Kif7</i>^<i>dd/dd</i> mutant mouse line. Images show Beta-galactosidase staining of tissue sections from <i>Gli1-lacZ;Kif7</i>^<i>dd/+</i> lungs and <i>Gli1-Lacz; Kif7</i>^<i>dd/dd</i> mutant lungs. Scale bar is 125 microns. (<b>G.</b>) Western blot analysis of protein lysates isolated from E18.5 control (+/+), <i>Kif7</i>^<i>dd/+</i> heterozygous, and <i>Kif7</i>^<i>dd/dd</i> homozygous mutant lungs. (<b>H.</b>) Western blot analysis of protein lysates isolated from postnatal mouse lung fibroblasts (MLFs) infected with control (scram) or <i>Kif7</i> gene specific shRNA expressing virus. Cells were infected, grown to confluence, and then serum starved for 3 days to allow for induction of <i>Hh</i> signaling. (<b>I.</b>) Immunoblots of nuclear fractions isolated from control and <i>Kif7</i> shRNA knock down mouse lung fibroblasts. Histone H3 (HH3) was used as nuclear fraction control, and a tubulin as a cytoplasmic control. (<b>J.</b>) Quantification of cell density following serum deprivation at 100% cell density. Control cells exit the cell cycle, while <i>Kif7</i> depleted MLFs re-enter the cell cycle and divide an additional time. N≥3, * P<0.05, **P<0.01.</p

KIF7 is a negative regulator of cell proliferation within the respiratory epithelium.

<p>(<b>A.-B’</b>) Transmission electron micrographs of E18.5 control and <i>Kif7</i>^<i>dd/dd</i> mutant respiratory airways. RBC (red blood cell) within the capillary adjacent to a type 1 AEC (1), AEC2 (2), Glycogen (G), Surfactant (S), Arrowheads denote lamellar bodies. (<b>C.-C’</b>) Immunofluorescent staining of SFTPC in E18.5 control and <i>Kif7</i>^<i>dd/dd</i> mutant lungs. (<b>D.-D’</b>) Co-immunoflorescent staining of Brdu (red) and CDH1 (green) in E17.5 control and <i>Kif7</i>^<i>dd/dd</i> mutant lungs. (<b>E.</b>) Quantification of relative number of immunopositive cells in control and <i>Kif7</i>^<i>dd/dd</i> mutant tissue sections. Immunopositive cells were counted and then divided by the total number of cells for control and mutant tissue sections. Control values were set as 1, and mutant values were divided by control values to provide a relative frequency of immunopositive cells per tissue section. At least 4 consecutive tissue sections were counted and averaged for an individual, from at least 4 sets of control and <i>Kif7</i>^<i>dd/dd</i> mutant lungs. * P<0.05, **P<0.01.</p

KIF7 Regulates cell proliferation and entry into S phase in mouse lung epithelial cells.

<p>(<b>A.</b>) Growth curve of control (Scram) and KIF7 depleted MLE15 cells. (<b>B.</b>) BrdU incorporation was measured by flow cytometry after release from G1 synchronization for control and KIF7 depleted cells. The analysis was performed using the BD BrdU-FitC kit and FlowJo software. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005525#pgen.1005525.s006" target="_blank">S6 Fig</a> for representative flow charts. (<b>C.</b>) Western blots of protein lysates from time course of serum starved G1 synchronized MLE15 cells (Time 0, and 8 or 10 hours after the re-addition of serum continuing media). (<b>D.</b>) Western blots of protein lysates from time course of G1 synchronized cells (Time 0) and following re-addition of serum containing media. (<b>E.+E’</b>) Immunostained tissue sections of control and <i>Kif7</i> mutant lungs. Cyclin d1 expression (brown) appears elevated in the <i>Kif7</i> mutant respiratory epithelium. Scale bar is 100 microns. N≥3, * P<0.05, **P<0.01.</p

KIF7 regulates mitotic exit in mouse lung epithelial cells.

<p>(<b>A.-D.</b>) Confocal immunofluorescent analysis of KIF7-GFP expression in different stages of the MLE15 cell cycle. <b>A.</b> G1 synchronized MLE15 cells expressing KIF7-GFP. <b>B.</b> A MLE15 cell in G1 with an unduplicated centrosome. <b>C</b>. A MLE15 cell in S/G2 with duplicated centrosomes, <b>D</b>. A MLE15 cell in mitosis. (<b>A.</b>) Confocal Z-stack of KIF7-GFP and B tubulin. Note that KIF7 localizes predominately to one side of the cell. Scale bar is 10 microns. (<b>B.+C.</b>) Co-immunofluorescent staining of KIF7-GFP with gamma (γ) tubulin, a marker for the centrosome. (<b>D.</b>) Co-immunofluorescent staining of KIF7-GFP colocalization with acetylated alpha tubulin, a marker of the spindle apparatus. Scale bar is 5 microns. (<b>E.</b>) Growth curve for KIF7-GFP overexpression in MLE15 cells. Cell proliferation was examined in GFP expressing and KIF7-GFP expressing MLE15 cells. (<b>F.</b>) Cell cycle profile for GFP, KIF7-GFP, and KIF7 L657*-GFP mutant expressing cells. (<b>G.</b>) FACs cell cycle analysis of <i>Kif7</i> overexpressing MLE15 cells. (<b>H.</b>) Western blots of protein lysates from GFP expressing (control) and Kif7-GFP expressing cells synchronized at the G2/M cell cycle checkpoint by nocodazole treatment. (<b>I.-J.</b>) Western blots of protein lysates from scram control and KIF7 depleted cells synchronized at the G2/M checkpoint with treatment with (NOC) nocodazole, TAME HCL (an anaphase promoting complex inhibitor), or nocodazole and TAME together. (<b>K.</b>) Flow cytometric analysis of DNA content following nocadazole washout for control and KIF7 depleted cells. KIF7 depleted cells routinely move from 4N to 2N faster than Scram controls. N≥3, * P<0.05, **P<0.01.</p

KIF7 regulates microtubule mstability in mouse lung epithelial (MLE15) cells.

<p>(<b>A.</b>) Growth curves of control (Scram) and KIF7 depleted cells following treatment and washout with the microtubule stabilizing drug, paclitaxel (TAX), and the microtubule depolymerizing drug, nocodazole (NOC). Cells were counted and plated, and then treated 24 hours later (Day 1). 18 hours later (Day 2), cells were washed 2 times in PBS, and then incubated with complete media. (<b>B.-C.</b>) Flow cytometric analysis of DNA content after overnight incubation with paclitaxel. (<b>D.+D’</b>) Confocal Z stacks of immunofluorescent staining of acetylated alpha tubulin in control and KIF7 depleted MLE15 cells. (<b>E.</b>) Western blot analysis of high passage asynchronous MLE15 cells. N≥3, * P<0.05, **P<0.01.</p