Mazhar, B. | bioRxiv Science

bioRxiv2020

OTX2 non-cell autonomous activity regulates inner retinal function

OTX2 is a homeoprotein transcription factor expressed in photoreceptors and bipolar cells in the retina. OTX2, like many other homeoproteins, transfers between cells and exerts non-cell autonomous effects such as promoting survival of retinal ganglion cells that do not express the protein. Here we used a genetic approach to target extracellular OTX2 in the retina by conditional expression of a secreted single chain anti-OTX2 antibody. Compared to control mice, the expression of this antibody by Parvalbumin-expressing neurons in the retina is followed by a reduction in visual acuity in one-month-old mice with no alteration of the retinal structure or cell type number or aspect. A- and b-waves measured by electroretinogram were also indistinguishable from control mice, suggesting no functional deficit of photoreceptors and bipolar cells. Mice expressing the OTX2-neutralizing antibody did show a significant doubling in the flicker amplitude, consistent with a change in inner retinal function. Our results show that interfering in vivo with OTX2 non-cell autonomous activity in the postnatal retina leads to an alteration in inner retinal cell functions and causes a deficit in visual acuity. Significance statementOTX2 is a homeoprotein transcription factor expressed in retinal photoreceptors and bipolar cells. Although the Otx2 locus is silent in the inner retina, the protein is detected in cells of the ganglion cell layer consistent with the ability of this class of proteins to transfer between cells. We expressed a secreted single chain antibody (scFv) against OTX2 in the retina to neutralize extracellular OTX2. Antibody expression leads to reduced visual acuity with no change in retinal structure, or photoreceptor or bipolar physiology; however, activity in the inner retina was altered. Thus, interfering with OTX2 non-cell autonomous activity in postnatal retina alters inner retinal function and causes vision loss, highlighting the physiological value of homeoprotein direct non-cell autonomous signaling.

neuroscience↗

bioRxiv2019

Vinculin targeting by Shigella IpaA promotes stable cell adhesion independent of mechanotransduction

The Shigella effector IpaA co-opts the focal adhesion protein vinculin to promote bacterial invasion. Here, we show that IpaA triggers an unreported mode of vinculin activation through the cooperative binding of its three vinculin-binding sites (VBSs) leading to vinculin oligomerization via its D1 and D2 head subdomains and highly stable adhesions resisting actin relaxing drugs. Using cross-linking mass spectrometry, we found that while IpaA VBSs1-2 bound to D1, IpaA VBS3 interacted with D2, a subdomain masked to other known VBSs. Structural modeling indicated that as opposed to canonical activation linked to interaction with D1, these combined VBSs interactions triggered major allosteric changes leading to D1D2 oligomerization. A cysteine-clamp preventing these changes and D1D2 oligomerization impaired growth of vinculin microclusters and cell adhesion. We propose that D1D2-mediated vinculin oligomerization occurs during the maturation of adhesion structures to enable the scaffolding of high-order vinculin complexes, and is triggered by Shigella IpaA to promote bacterial invasion in the absence of mechanotransduction. SummaryThe Shigella IpaA effector binds to cryptic vinculin sites leading to oligomerization via its head domain. This vinculin oligomerization mode appears required for the maturation and strengthening of cell adhesion but is co-opted by invasive bacteria independent of actomyosin contractility.

microbiology↗