The localization of chitin synthase mediates the patterned deposition of chitin in developing Drosophila bristles

Abstract

28The insect exoskeleton is a morphologically complex structure that is a key for the life style of this very 29 successful group of animals. The cuticular cytoskeleton contains proteins, lipids and the N-acetyl 30 glucosamine polymer chitin. Chitin is a highly patterned and essential component of the insect 31 exoskeleton synthesized by chitin synthase. In most body regions chitin fibrils are found in a stack of 32 parallel arrays that can be detected by transmission electron microscopy. Each array is rotated with 33 respect to the layers above and below. In sensory bristles, chitin primarily accumulates in bands parallel 34 to the proximal/distal axis of the bristle. These bands are visible by confocal microscopy providing 35 experimental advantages. We have used this cell type and an edited chitin synthase gene to establish 36 that the bands of chitin are closely associated with stripes of chitin synthase. This argues that the 37 localization of chitin synthase plays an important role in mediating the patterned deposition of chitin in 38 insect cuticle. However, other data suggest this connection may not be absolute. Several genes are 39 essential for proper chitin deposition. We found one of these, Rab11 is required for the insertion of 40 chitin synthase into the plasma membrane and a second, duskylike is required for plasma membrane 41 chitin synthase to properly localize into stripes. We also established that the actin cytoskeleton is 42 required for the proper localization of chitin synthase and chitin in developing sensory bristles. An 43 unexpected finding is that chitin synthase and other membrane proteins are shed during or after the 44 process of cuticle deposition and this may explain cases where there is a lack of a sharp phenotypic 45 boundary between cells that have or lack chitin synthase activity.

Short distance non-autonomy and intercellular transfer of chitin synthase in Drosophila

“…Flies homozygous for each of the edits were viable and showed no dramatic 141 phenotype when examined in a stereomicroscope. However, when we mounted wings for examination 142 in the compound microscope we observed thin, bent and generally deformed wing hairs (trichomes) in 143 the smFP homozygotes but not the NG homozygotes (Adler, 2020). We further established that the 144 Kkv::smFP protein accumulated to a much lower level than Kkv::NG.…”

“…All of the experiments where we detected puncta in living pupae required imaging of the Kkv::NG fusion 245 protein. Experiments described earlier established that NG was a valid reporter for Kkv in bristles so it 246 seemed likely that it was also a valid reporter for Kkv in puncta (Adler, 2020). To test this hypothesis I 247 immunostained pupal cuticle using both anti-NG and anti-Kkv-M antibodies.…”

“…When we examined kkv::smFP/Df 145 flies the hair phenotype appeared slightly stronger. These two observations let me to conclude 146 kkv::smFP was a hypomorph due to the protein not accumulating efficiently (Adler, 2020). I 147 subsequently noticed that a minority of the kkv::smFP/Df flies had deformed wings soon after or at 148 eclosion ( Fig 3).…”

“…That there is a single chitin synthase makes it an attractive 73 target for studying details of cuticle deposition. I recently found that an edited kkv which fused an smFP 74 tag to the c terminal end of the protein resulted in a viable hypomorphic allele (Adler, 2020). This was 75 identified on the basis of a phenotype in the hairs that cover the wing.…”

“…As described previously I used Crispr/Cas9 to tag the endogenous kkv gene with either Neon Green or 140 smFP (Adler, 2020). Flies homozygous for each of the edits were viable and showed no dramatic 141 phenotype when examined in a stereomicroscope.…”