Loss of function of Synj2 leads to cochlear hair cell loss.

<p>Ultrastructural analysis using scanning electron microscopy of cochlear hair cells in <i>Synj2</i>^N538K/N538K and <i>Synj2</i>^+/+ mice. Apical, mid and basal cochlear turns were examined at 2, 4, 8 and 12 weeks of age. As early as 2 wks of age, signs of hair cell degeneration are seen in outer hair cells of the basal cochlear turn (Panel D). At 4 weeks, the majority of basal outer hair cells appear sunken with fused stereocilia (Panel H). Mid cochlear inner hair cells show stereocilia fusion at 8 weeks (Panel L). Fused stereocilia or complete loss of hair bundles is common in mid region outer hair cells at 12 weeks (Panel P). Panel T shows the structure of a normal outer hair cell. OHC, outer hair cells; IHC, inner hair cells. Scale bar: 4 µm.</p

The N538K mutation abolishes Synj2 inositol polyphosphate 5-phosphatase activity.

<p>Domain structure of Synj2 indicating the position of the N538K mutation in the 5-phosphatase domain (Panel A). The phosphoinositide substrates hydrolyzed by the Sac1 and 5-phosphatase domains are indicated. Protein sequence alignment of murine 5-phosphatases showing N⁵³⁸ (boxed) is conserved in all 10 mammalian 5-phosphatases (Panel B). Accession numbers: Synj2: Q9D2G5, Synj1: NP_001157955, Type I: AAH56341, Type II: CAM16097, SHIP1: Q9ES52, SHIP2: Q6P549, PIPP: AAI31635, INPP5E: NP_149125, SKIP: NP_032942, OCRL: NP_796189. GST-Synj2 wild-type or Synj2^N538K mutant 5-phosphatase domains or GST alone were purified from <i>E. coli</i> and assayed for PtdIns([³²P]3,4,5)P₃ 5-phosphatase activity (Panel C). Lipid products were separated by thin layer chromatography (top left panel). The migration of PtdIns(3,4)P₂ and PtdIns(3,4,5)P₃ are indicated. The relative amount of recombinant protein added to each reaction was determined by immunoblotting with GST antibodies (lower left panel). The relative percentage of PtdIns(3,4,5)P₃ substrate hydrolyzed was determined by densitometry (right panel). Bars represent mean ± SEM from 3 independent experiments. **p&lt;0.001. Recombinant GST-Synj2 wild-type or Synj2^N538K mutant 5-phosphatase domains or GST alone were purified from <i>E. coli</i> and assayed for PtdIns(4,5)P₂ 5-phosphatase activity (Panel D). Phosphate release (pmol/25 µl sample) was measured using a malachite green assay (left panel). Bars represent mean ± SEM from 2 independent experiments. The relative amount of recombinant protein added to each reaction was determined by Western blotting with GST antibodies (right panel). **p&lt;0.001.</p

<i>Synj2</i>^N538K/N538K mutant mice show progressive age-related hearing loss.

<p>Hearing thresholds (dB SPL) in <i>Synj2</i>^+/+, <i>Synj2</i>^N538K/+ and <i>Synj2</i>^N538K/N538K mice at age 4, 8, 12 and 24 weeks. Hearing threshold values shown are averages of at least 20 mice per genotype and error bars represent the standard deviation.</p

Synj2^N538K/N538K cochleae exhibit degeneration.

<p>Synj2^+/+ (Panels A, B and C) and Synj2^N538K/N538K (Panels D, E and F) cochleae were sectioned at 4 (Panels A and D), 8 (Panels B and E) and 12 (Panels C and F) weeks of age and H&amp;E stained. Representative cochleae are shown. Synj2^N538K/N538K cochleae show signs of degeneration at 8 and 12 weeks of age, with evidence of collapse of the organ of Corti (arrow head, panels E and F) and degeneration of spiral ganglion neurons (arrow, panels E and F). SG, spiral ganglion; OC, organ of corti; OHC, outer hair cells; IHC, inner hair cell. Scale bar: 100 µm.</p

Optimizing metastatic-cascade-dependent Rac1 targeting in breast cancer: Guidance using optical window intravital FRET imaging

Assessing drug response within live native tissue provides increased fidelity with regards to optimizing efficacy while minimizing off-target effects. Here, using longitudinal intravital imaging of a Rac1-Forster resonance energy transfer (FRET) biosensor mouse coupled with in vivo photoswitching to track intratumoral movement, we help guide treatment scheduling in a live breast cancer setting to impair metastatic progression. We uncover altered Rac1 activity at the center versus invasive border of tumors and demonstrate enhanced Rac1 activity of cells in close proximity to live tumor vasculature using optical window imaging. We further reveal that Rac1 inhibition can enhance tumor cell vulnerability to fluid-flow-induced shear stress and therefore improves overall anti-metastatic response to therapy during transit to secondary sites such as the lung. Collectively, this study demonstrates the utility of single-cell intravital imaging in vivo to demonstrate that Rac1 inhibition can reduce tumor progression and metastases in an autochthonous setting to improve overall survival. </p