LRCH proteins: a novel family of cytoskeletal regulators

Abstract

Background: Comparative genomics has revealed an unexpected level of conservation for gene products across the evolution of animal species. However, the molecular function of only a few proteins has been investigated experimentally, and the role of many animal proteins still remains unknown. Here we report the characterization of a novel family of evolutionary conserved proteins, which display specific features of cytoskeletal scaffolding proteins, referred to as LRCHs.

Principal findings: Taking advantage of the existence of a single LRCH gene in flies, dLRCH, we explored its function in cultured cells, and show that dLRCH act to stabilize the cell cortex during cell division. dLRCH depletion leads to ectopic cortical blebs and alters positioning of the mitotic spindle. We further examined the consequences of dLRCH deletion throughout development and adult life. Although dLRCH is not essential for cell division in vivo, flies lacking dLRCH display a reduced fertility and fitness, particularly when raised at extreme temperatures.

Conclusion/significance: These results support the idea that some cytoskeletal regulators are important to buffer environmental variations and ensure the proper execution of basic cellular processes, such as the control of cell shape, under environmental variations.

Figure 1. dLRCH defines a novel protein family evolutionary conserved in animals.

A. The predicted Drosophila dLRCH protein includes two regions with recognizable motifs: eight repetitions of Leucine Rich Repeats (LRR) from position 92 to 289 and a Calponin Homology (CH) domain in the C-terminus (position 668 to 771). Domains that display a typical LRR organization are shown as LRR-labeled green boxes, degenerate LRR-like structure are shown as green boxes. B. Number of proteins that contain LRR, CH or LRR+CH domains in flies and humans. C. Distribution of LRCH proteins throughout different animal phyla. Hs, Homo sapiens; Mm, Mus musculus; Gg, Gallus gallus; Fr, Fugu rubripes; Ce, Caenorhabditis elegans; Cb, Caenorhabditis briggsae; Dm, Drosophila melanogaster; Ag, Anopheles gambiae; Ci, Ciona intestinalis.

Figure 2. dLRCH accumulates at the mitotic cortex and cleavage furrow.

Sub-cellular distribution of a GFP-dLRCH fusion protein throughout the successive stages of cell division, as assayed in cultured Drosophila S2 cells. (a–c″) Co-detection of GFP-dLRCH (green) and F-actin (red), from metaphase to telophase. In metaphase, dLRCH is enriched at the cell cortex, co-localizing with F-actin. Starting from anaphase, GFP-dLRCH accumulates at the cleavage furrow (arrows). During late telophase, GFP-dLRCH becomes concentrated at the midbody region (arrowhead) as shown in double labelings with α-tubulin (d–d″). DNA is in blue in merged images (a″, b″, c″ and d″).

Figure 3. dLRCH parallels the distribution of activated Moe during cell division.

The GFP-dLRCH fusion protein co-localizes with activated Moe during mitosis. Distribution of P-Moe (red) in dividing S2 cells shows a pattern reminiscent to GFP-dLRCH localization (green). Whereas in metaphase both proteins are detected around the entire cell cortex, GFP-dLRCH and P-Moe become restricted to the cleavage furrow (arrows) from anaphase to the end of mitosis. DNA is in blue.

Figure 4. dLRCH depletion induces deformation of the mitotic cortex.

A. Time-lapse frames of living S2 cells stably expressing Tubulin-GFP. Top panels shows a dividing S2 cell in control conditions (no dsRNA), lower panels shows a dLRCH-depleted cell displaying abnormal cortical protrusions (arrows) from pro/metaphase to ana/telophase. B. Quantification of the defects observed in cortical and spindle organization upon depletion of dLRCH and/or Moe. The left chart plots cortical deformation, with the presence of at least one bleb during cell division (with the exception of those normally observed at the polar cortex). Right panel shows the variation in spindle orientation after treatment with dLRCH, Moe or both dsRNA. Spindle rotation was estimated by measuring the angle of the axis of the spindle from the first metaphase observed to that of telophase (n = 329, 391, 280 and 182 cells for control, Moe dsRNA, dLRCH dsRNA and Moe+dLRCH dsRNA, respectively). Errors bars represent SD.

Figure 5. Expression pattern of dLRCH during Drosophila embryogenesis.

Embryonic expression of dLRCH is ubiquitous and reinforced in specific tissues, as shown by whole mount in situ hybridization to dLRCH mRNA. (a–h, e′) Pictures correspond to lateral views of embryos from early to late embryonic stages or (f′–h′) to dorsal views of embryos from stage 13 to 16. Besides a low-level ubiquitous expression, higher levels of dLRCH mRNA accumulates in tracheal pits (arrowheads in panels c and e′), the central nervous system (arrows in panels e, f, g, h, brackets in f′ and g′), as well as in embryonic gonads (stars in panels g, h and h′). (d) The picture shows the staining obtained with a control sense probe.

Figure 6. Consequences of the lack of dLRCH on development and lifespan.

A. Genomic organization of the dLRCH (CG6860) locus, with coding exons drawn in light blue. The Df(2L)dLRCH was generated by provoking recombination between two transposons carrying FRT sites (Pbac(WH)f01198 and P(XP)CG6860[d04045]) and its molecular structure was verified by PCR. Df(2L)dLRCH removes most dLRCH coding sequence, but the first exon which encodes 101 aa. The 5′ breakpoint of Df(2L)dLRCH is located 1.8 kb upstream of CLIP-190 and thus not likely to interfere with CLIP-190 expression, although the latter point remains to be tested experimentally. B. Survival rate of Df(2L)dLRCH homozygous individuals throughout development, at 25°C (left panel, n = 50 embryos), 17°C or 29°C (right panel, n = 50 and 60, respectively). The parental line P[XP]d04045 was used as control. C. The fertility was evaluated as the number of adult progeny emerging from crosses between a control w strain and Df(2L)dLRCH mutants (n = 872, 575, 0 for control females, heterozygous and homozygous Df(2L)dLRCH; and n = 872, 808 and 752 for males of the corresponding genotypes). All values were compared to control crosses (100%). Errors bars represent SD. D. Longevity of adult flies submitted to 25°C conditions. n = 160 for control females, n = 161 for control males, n = 106 for Df(2L)dLRCH heterozygous females, n = 93 for Df(2L)dLRCH heterozygous males, n = 87 for Df(2L)dLRCH homozygous females and n = 72 for Df(2L)dLRCH homozygous males.