Functional assessment of lysosomal Rab7 and RILP with RNA interference and overexpression in Spodoptera frugiperda Sf9 cell lines.

The interaction manner and biological function of Rab7 and its effector, Rab-interacting lysosomal protein (RILP), remain unclear in invertebrates. We provide a protocol for detecting the effects of Rab7 and RILP terminals on lysosome and autophagy in Spodoptera frugiperda Sf9 cells with overexpression and RNA interference. We describe steps for overexpressing plasmids, generating long double-stranded RNA, and transfecting them into Sf9 cells. We then detail procedures for cell immunofluorescence imaging with harmine treatment and fluorescence analysis. For complete details on the use and execution of this protocol, please refer to Cui et al. (2023).1.

Hydroxylated sphingolipid biosynthesis regulates photoreceptor apical domain morphogenesis.

Apical domains of epithelial cells often undergo dramatic changes during morphogenesis to form specialized structures, such as microvilli. Here, we addressed the role of lipids during morphogenesis of the rhabdomere, the microvilli-based photosensitive organelle of Drosophila photoreceptor cells. Shotgun lipidomics analysis performed on mutant alleles of the polarity regulator crumbs, exhibiting varying rhabdomeric growth defects, revealed a correlation between increased abundance of hydroxylated sphingolipids and abnormal rhabdomeric growth. This could be attributed to an up-regulation of fatty acid hydroxylase transcription. Indeed, direct genetic perturbation of the hydroxylated sphingolipid metabolism modulated rhabdomere growth in a crumbs mutant background. One of the pathways targeted by sphingolipid metabolism turned out to be the secretory route of newly synthesized Rhodopsin, a major rhabdomeric protein. In particular, altered biosynthesis of hydroxylated sphingolipids impaired apical trafficking via Rab11, and thus apical membrane growth. The intersection of lipid metabolic pathways with apical domain growth provides a new facet to our understanding of apical growth during morphogenesis.

Peroxisome Elevation Induces Stem Cell Differentiation and Intestinal Epithelial Repair.

Epithelial-repair-dependent mucosal healing (MH) is associated with a more favorable prognosis for patients with inflammatory bowel disease (IBD). MH is accomplished via repair and regeneration of the intestinal epithelium. However, the mechanism underlying MH is ill defined. We found a striking upregulation of peroxisomes in the injured crypts of IBD patients. By increasing peroxisome levels in Drosophila midguts, we found that peroxisome elevation enhanced RAB7-dependent late endosome maturation, which then promoted stem and/or progenitor-cell differentiation via modulation of Janus Kinase (JAK) and Signal Transducer and Activator of Transcription (STAT)-SOX21A signaling. This in turn enhanced ISC-mediated regeneration. Importantly, RAB7 and SOX21 were upregulated in the crypts of IBD patients. Moreover, administration of drugs that increased peroxisome levels reversed the symptoms of dextran sulfate sodium (DSS)-induced colitis in mice. This study demonstrates a peroxisome-mediated epithelial repair mechanism, which opens a therapeutic avenue for the enhancement of MH in IBD patients.

Statistical analysis of particle trajectories in living cells.

Recent advances in molecular biology and fluorescence microscopy imaging have made possible the inference of the dynamics of molecules in living cells. Such inference allows us to understand and determine the organization and function of the cell. The trajectories of particles (e.g., biomolecules) in living cells, computed with the help of object tracking methods, can be modeled with diffusion processes. Three types of diffusion are considered: (i) free diffusion, (ii) subdiffusion, and (iii) superdiffusion. The mean-square displacement (MSD) is generally used to discriminate the three types of particle dynamics. We propose here a nonparametric three-decision test as an alternative to the MSD method. The rejection of the null hypothesis, i.e., free diffusion, is accompanied by claims of the direction of the alternative (subdiffusion or superdiffusion). We study the asymptotic behavior of the test statistic under the null hypothesis and under parametric alternatives which are currently considered in the biophysics literature. In addition, we adapt the multiple-testing procedure of Benjamini and Hochberg to fit with the three-decision-test setting, in order to apply the test procedure to a collection of independent trajectories. The performance of our procedure is much better than the MSD method as confirmed by Monte Carlo experiments. The method is demonstrated on real data sets corresponding to protein dynamics observed in fluorescence microscopy.

Assessment of chloroquine treatment for modulating autophagy flux in brain of WT and HD mice.

BACKGROUND: Increasing mutant huntingtin (mHTT) clearance through the autophagy pathway may be a way to treat Huntington's disease (HD). Tools to manipulate and measure autophagy flux in brain in vivo are not well established. OBJECTIVE: To examine the in vivo pharmacokinetics and pharmacodynamics of the lysosomal inhibitor chloroquine (CQ) and the levels of selected autophagy markers to determine usefulness of CQ as a tool to study autophagy flux in brain. METHODS: Intraperitoneal injections of CQ were administered to WT and HD(Q175/Q175) mice. CQ levels were measured by LC-MS/MS in WT brain, muscle and blood at 4 to 24 hours after the last dose. Two methods of tissue preparation were used to detect by Western blot levels of the macroautophagy markers LC3 II and p62, the chaperone mediated autophagy receptor LAMP-2A and the late endosome/lysosomal marker RAB7. RESULTS: Following peripheral administration, CQ levels were highest in muscle and declined rapidly between 4 and 24 hours. In the brain, CQ levels were greater in the cortex than striatum, and levels persisted up to 24 hours post-injection. CQ treatment induced changes in LC3 II and p62 that were variable across regions and tissue preparations. HD(Q175/Q175) mice exposed to CQ had variable but diminished levels of LC3 II, p62 and LAMP-2A, and increased levels of RAB7. Higher levels of mHTT were found in the membrane compartment of CQ treated HD mice. CONCLUSION: Our findings suggest that the response of brain to CQ treatment, a blocker of autophagy flux, is variable and not as robust as it has been demonstrated in vitro, suggesting that CQ treatment has limitations for modulating autophagy flux in vivo. Alternative methods, compounds, and technologies need to be developed to further investigate autophagy flux in vivo, especially in the brain.