Visualizing the membrane dynamics of photoreceptor neurons
To visualize the process of protein transport and membrane renewal, the group developed the photoconversion technique which enables visualization of protein transport. This photoconversion technique allows enhancing the contrast for newly synthesized proteins by a factor of 1000 – 4000 fold. The technique was combined with high-resolution confocal microscopy to study the ciliary transport of rod photoreceptor membrane proteins. Disruption of ciliary transport mechanism is the major cause of inherited photoreceptor degeneration.
With photoconversion and other techniques,the group found that rhodopsin and peripherin/rds, two major OS constituents,are transported by distinct mechanisms (Tian et al. 2014). Rhodopsin is trafficked through a conventional secretory pathway, whereas peripherin/rds is trafficked through an unconventional secretory pathway. We also found that different combinations of trafficking signals allow these proteins to select specific and distinct routes of trafficking (Lodowski et al. 2013 and Tian et al. 2014). When trafficking signals become mis-coordinated, rhodopsin mislocalizes to the plasma membrane where rhodopsin exerts its toxic effects (Ropelewski and Imanishi 2019). This mislocalization is of clinical importance, as it is observed in the retina of most patients with retinitis pigmentosa. Imanishi Lab is specialized to characterize the mechanisms of membrane protein transport and renewal in photoreceptor cells.
Discovering Therapy for Sensorineural Disorders
Missense mutations are major causes of inherited disorders.Imanishi lab invented a novel dual-reporter assay for screening small moleculeswhich can potentially ameliorate such disease conditions. This assay involvesfluorescently labeling the target protein in one color (e.g. green or yellow)while simultaneously introducing a proteasome-targeted fluorescent protein(DsRed-Express-DR) in order to determine if the small molecule treatment istargeting the proteasome or stabilizing the target proteins by other mechanisms. If drug treatment causes both greenfluorescent protein and red fluorescent protein accumulation, then it is likelya general inhibitor of the proteasome. However, if red protein fluorescencedoes not increase but green fluorescence increases, then the drug treatment isspecific to the target protein and not a general proteasome inhibitor. Weutilized this screening strategy to discover novel small molecules whichimproved the stability of Clarin-1 mutant associated with Usher syndrome typeIII. One of the molecules delayed the onset of hearing loss in a mouse model ofUsher syndrome type III (Alagramam et al. 2016).