Mutations in LOXHD1, an Evolutionarily Conserved Stereociliary Protein, Disrupt Hair Cell Function in Mice and Cause Progressive Hearing Loss in Humans

Hearing loss is the most common form of sensory impairment in humans and is frequently progressive in nature. Here we link a previously uncharacterized gene to hearing impairment in mice and humans. We show that hearing loss in the ethylnitrosourea (ENU)-induced samba mouse line is caused by a mutation in Loxhd1. LOXHD1 consists entirely of PLAT (polycystin/lipoxygenase/α-toxin) domains and is expressed along the membrane of mature hair cell stereocilia. Stereociliary development is unaffected in samba mice, but hair cell function is perturbed and hair cells eventually degenerate. Based on the studies in mice, we screened DNA from human families segregating deafness and identified a mutation in LOXHD1, which causes DFNB77, a progressive form of autosomal-recessive nonsyndromic hearing loss (ARNSHL). LOXHD1, MYO3a, and PJVK are the only human genes to date linked to progressive ARNSHL. These three genes are required for hair cell function, suggesting that age-dependent hair cell failure is a common mechanism for progressive ARNSHL

The Role and Mechanisms of Dlg5 in the Regulation of the Hippo Signaling Pathway

Thesis (Ph.D.)--University of Washington, 2015The Hippo signal transduction pathway plays a pivotal role in regulation of normal development, adult organ homeostasis, and cancer. At the core of the Hippo pathway, MST1/2 kinases phosphorylate and activate LATS1/2 kinases, which in turn phosphorylate and inactivate transcriptional co-activators YAP1 and TAZ. Phosphorylated YAP1 and TAZ are inhibited from entering the nucleus and are instead cytoplasmically retained or degraded. Cells monitor their microenvironment, by sensing local cell density and cellular polarity, and use Hippo signaling to regulate the maintenance of normal cell numbers necessary for tissue function. The mechanisms responsible for the connection between the cellular microenvironment and Hippo pathway are poorly understood. In this study, in collaboration with Dr. Andrew Emili's laboratory in the University of Toronto, I reveal a novel mechanistic connection between the Hippo signaling pathway and the apical-basal cell polarity protein DLG5. I found that DLG5 binds to MST1/2 kinases of the core Hippo pathway. Inactivation of Dlg5 in mice in vivo and in primary cells ex vivo results in increased Hippo pathway activity and decreased levels and activity of YAP1 and TAZ. Overexpression of DLG5 inhibits Hippo signaling and promotes the expression of Hippo pathway target genes. In genetic epistasis experiments in mice, Dlg5 shows a strong genetic interaction with both Yap1 and Taz (Wwtr1). Mechanistically, I found that DLG5 negatively regulates the Hippo pathway by inhibiting the binding between MST1/2 and LATS1/2. This study increases the understanding of the connection between apical-basal polarity and the Hippo pathway and identifies apical-basal polarity family protein DLG5 as a novel interactor and regulator of the Hippo signal transduction pathway

The Spindle Checkpoint Functions of Mad3 and Mad2 Depend on a Mad3 KEN Box-mediated Interaction with Cdc20-Anaphase-promoting Complex (APC/C)*S⃞♦

Mitotic progression is driven by proteolytic destruction of securin and cyclins. These proteins are labeled for destruction by an ubiquitin-protein isopeptide ligase (E3) known as the anaphase-promoting complex or cyclosome (APC/C). The APC/C requires activators (Cdc20 or Cdh1) to efficiently recognize its substrates, which are specified by destruction (D box) and/or KEN box signals. The spindle assembly checkpoint responds to unattached kinetochores and to kinetochores lacking tension, both of which reflect incomplete biorientation of chromosomes, by delaying the onset of anaphase. It does this by inhibiting Cdc20-APC/C. Certain checkpoint proteins interact directly with Cdc20, but it remains unclear how the checkpoint acts to efficiently inhibit Cdc20-APC/C activity. In the fission yeast, Schizosaccharomyces pombe, we find that the Mad3 and Mad2 spindle checkpoint proteins interact stably with the APC/C in mitosis. Mad3 contains two KEN boxes, conserved from yeast Mad3 to human BubR1, and mutation of either of these abrogates the spindle checkpoint. Strikingly, mutation of the N-terminal KEN box abolishes incorporation of Mad3 into the mitotic checkpoint complex (Mad3-Mad2-Slp1 in S. pombe, where Slp1 is the Cdc20 homolog that we will refer to as Cdc20 hereafter) and stable association of both Mad3 and Mad2 with the APC/C. Our findings demonstrate that this Mad3 KEN box is a critical mediator of Cdc20-APC/C inhibition, without which neither Mad3 nor Mad2 can associate with the APC/C or inhibit anaphase onset