Tight junctions prevent paracellular flow and maintain cell polarity in an epithelium. These junctions are also required for maintaining the blood-testis barrier, which is essential for sperm differentiation. Septate junctions in insects are orthologous to the tight junctions. In Drosophila testis, major septate junction components co-localize at the interface of germline and somatic cells initially, and then condense between the two somatic cells in a cyst after germline meiosis. Their localization is extensively remodeled in subsequent stages. We find that characteristic septate junctions are formed between the somatic cyst cells at the elongated spermatid stage. Consistent with previous reports, knockdown of essential junctional components – Discs-large-1 and Neurexin-IV – during the early stages disrupted sperm differentiation beyond the spermatocyte stage. Knockdown of these proteins during the final stages of spermatid maturation caused premature release of spermatids inside the testes, resulting in partial loss of male fertility. These results indicate the importance of maintaining the integrity of the somatic enclosure during spermatid coiling and release in Drosophila testis. It also highlights the functional similarity with the tight junction proteins during mammalian spermatogenesis.

Highlights d Amphiphysin recruits Rho1 on somatic cell membrane wrapped around spermatid heads d Rho1 triggers F-actin assembly through the formins Diaphanous and DAAM d Rho1-Rok assembles an actomyosin scaffold around the folded plasma membrane d The actomyosin scaffold clamps spermatid heads together into a tight bundle

statement: Dubey et al., showed that septate junctions stitch the somatic 15 enclosure around maturing spermatids in Drosophila testis. Maintaining the integrity of 16 this junction is essential for proper release of spermatids. Abstract 20Tight junctions prevent the paracellular flow and maintain cell polarity in an epithelium. 21These are also essential for maintaining the blood-testis-barrier involved in regulating 22 sperm differentiation. Septate junctions are orthologous to the tight junctions in insects. 23In Drosophila testis, major septate junction components co-localize at the interface of 24 germline and somatic cells initially and then condense between the two somatic cells in 25 a cyst after germline meiosis. Their localization is extensively remodeled in subsequent 26 stages. We find that characteristic septate junctions are formed between the somatic cyst 27 cells at the elongated spermatid stage. Consistent with the previous reports, knockdown 28 of essential junctional components, Discs-large-1 and Neurexin-IV, in the somatic cyst 29 cells, during the early stages, disrupted sperm differentiation beyond the spermatocyte 30 stage. Somatic knockdown of these proteins during the final stages of spermatid 31 maturation caused premature release of spermatids inside the testes, resulting in partial 32 loss of male fertility. These results indicate the importance of maintaining mechanical 33 integrity of the somatic enclosure during spermatid coiling and release in Drosophila 34 testis. It also highlights the functional similarity with the tight junction proteins during 35 spermatogenesis in mammalian testes.36 37

Summary Here we describe a simple step-by-step protocol for collecting high-resolution, time-lapse images of intact Drosophila testis ex vivo for a limited period using a confocal microscope, with minimum photo-toxic damage, to monitor spermatid individualization, coiling, and release. The F-actin dynamics during spermatid morphogenesis can be further investigated through laser ablations, Fluorescence-Recovery-After-Photobleaching, and drug treatments, using this protocol. For complete details on the use and execution of this protocol, please refer to Dubey et al. (2016) , Dubey et al. (2019) , and Kapoor et al. (2021) .

Nanoscale bending of plasma membrane increases cell adhesion, induces cell-signalling, triggers F-actin assembly and endocytosis in tissue-cultured cells. The underlying mechanisms are not very well understood. Here, we show that stretching the plasma membrane of somatic cyst cell around rigid spermatid heads generates a stable, tubular endomembrane scaffold supported by contractile actomyosin. The structure resembles an actin-basket covering the bundle of spermatid heads. Genetic analysis suggests that the actomyosin organisation is nucleated exclusively by the Formins, Diaphanous and DAAM, downstream of Rho1, recruited by the Bin-Amphyphysin-Rvs (BAR)-domain protein, Amphiphysin, around the spermatid heads. Actomyosin activity at the actin-basket gathers the spermatid heads into a compact bundle and resists the invasion of the somatic cell by the intruding spermatids. These observations reveal a new response mechanism of nanoscale bending of the plasma membrane, which generates a novel cell adhesion strategy through active clamping.