Background

Cytokinesis is a final step of cell division wherein parent cell cytoplasmic contents and plasma membrane are physically divided between the two daughter cells. In animal cells, immediately after chromosomes are segregated to the poles, the spindle-mediated mechanisms determine the site of actomyosin ring assembly at the cell cortex. Subsequently, the actomyosin contractile ring that anchors the plasma membrane, and the central spindle begins to constrict, leading to the generation of cleavage furrow (Figure 1B). In mitotic cells, the plasma membrane between two daughter cells is pinched off in a process known as abscission, leading to the physical separation of daughter cells. However, unlike mitotic cells, in male germ cells abscission does not occur at the end of cytokinesis. As a result, all the daughter cells originating from the gonioblast are interconnected via their cytoplasmic bridges, known as ring canals. It is thought that the ring canals play an important role in intercellular communication and signaling [1]. All the cells in a cyst connected via cytoplasmic bridges undergo synchronous divisions [2, 3] (Video 1).

Drosophila melanogaster has been shown to be a valuable genetic model to study cytokinesis in vivo. Conventionally, Drosophila embryos undergoing cellularization [4, 5], proliferating larval neuroblasts [6, 7], and male meiotic spermatocytes [8, 9] have been used in cytokinesis studies. Meiotic spermatocytes in Drosophila testes offer several unique advantages over other systems to study cytokinesis. These include the relatively large size of meiotic spindle, weak spindle checkpoint, unambiguous identification of cytokinetic mutants due to characteristic morphological differences, and finally, live imaging of spermatocytes in isolated cysts or intact pupal testes. The relatively large size of male meiotic spindle is a favorable cytological feature in spermatocytes that allows precise localization of many cytokinetic proteins including microtubule and contractile ring–associated proteins. Male meiotic cells also show prominent central spindle (Figure 1B) (a bundle of antiparallel, interdigitating microtubules localizes to the center of segregating chromosomes where the assembly of contractile ring begins) [10, 11]. In neuroblasts, the presence of an abnormal spindle activates spindle checkpoint, which in turn arrests the cells in metaphase and precludes the observation of subsequent steps in cell division including cytokinesis [11–14]. However, due to weak spindle checkpoint, spermatocytes continue through the cell division stages despite the abnormal spindle. Thus, using meiotic cytokinesis, it is possible to ask whether a gene involved in spindle formation also has a role in later stages of cell division including cytokinesis. For instance, it was shown that mutations in the polo and abnormal spindle (asp) genes cause metaphase arrest in larval neuroblasts but produce frequent cytokinetic failures in spermatocytes [12–14]. Drosophila male meiosis is also an excellent model for cytokinesis due to the unique organization of mitochondria in round spermatids. During male meiotic cytokinesis, mitochondria is equally partitioned between the two daughter cells; immediately after cytokinesis, it aggregates to form a phase-dark structure known as Nebenkern. Failure in cytokinesis abrogates mitochondrial segregation; as a result, round spermatids show unusually large mitochondria associated with multiple nucleus (Figure S1) [15]. Thus, the presence of unusually large mitochondria with multiple nucleus is a characteristic feature of meiotic cytokinesis in spermatocytes (Figure S1) [3, 15].

Figure 1. Male meiotic cytokinesis in adult Drosophila testis. (A) Cartoon representing dissected adult testis showing various stages of germ cell differentiation. Drosophila spermatogenesis shares a significant amount of similarity with the mammalian system. In many species, including Drosophila melanogaster, male germ cell differentiation is a sequential process. It begins with the germline stem cell (GSC) attached to the hub cells at the tip of the testis undergoing asymmetric division to yield one committed progenitor called gonioblast (GB). Two cyst stem cells (CySCs) that surround the GSC also undergo asymmetric division to produce two cyst cells that encase the GB. Subsequently, the GB undergoes four rounds of mitotic divisions, resulting in the 16-cell stage known as spermatogonia (SG). With the premeiotic S phase and an impressive growth during G2 phase due to robust transcriptional activity, spermatogonia become spermatocytes (SC), which in turn synchronously undergo two rounds of meiotic divisions (meiosis I & meiosis II) resulting in the haploid, 64-cell stage known as round spermatids (RS). Due to a lack of abscission step at the end of cytokinesis in germ cells, all cell divisions originating from a single GB are connected to each other via cytoplasmic bridges known as ring canals (RCs). After completion of meiosis, all the mitochondria in individual spermatids aggregate around the basal body at one end of the nucleus to form a unique structure called nebenkern. Subsequently, round spermatids elongate via complex morphological changes including polarization of spermatid nucleus, dramatic nuclear size reduction due to histamine to protamine switch, elongation of nebenkern, axoneme growth, and formation of acrosome, a membrane-bound organelle required for fertilization. Finally, the individual sperms are physically separated by a complex process known as individualization, wherein actin-based cones migrate from head to tail fashion with the concomitant removal of cytoplasmic contents [cystic bulge (CB) and waste bag (WB)] and wrapping each spermatozoon with its own plasma membrane. (B) Representative live images showing localization of central spindle–associated protein feo (fascetto tagged with mCherry) (left panel) and a plasma membrane–associated protein tGPH (PH domain of Grp1fused to GFP) (middle panel), during cytokinesis. Right panel shows the merge of both channels and arrow indicates the site of cleavage furrow.

Several excellent protocols have been described in the literature for the isolation of Drosophila testes from embryo to adult developmental stages and ex vivo imaging of whole mount pupal testes and isolated spermatocyte cysts [16–22]. However, detailed protocols focused on live imaging of male meiotic cytokinesis in dissected germ line cysts have not been described. Further, live imaging of male meiotic cytokinesis in whole mount adult testes has not been demonstrated. The protocols described here build upon the methods developed by Gartner et al. [18] and Karabasheva, G. and Smyth, J.T. [19], extending their application to male meiotic cytokinesis in adult Drosophila testes. Gartner et al. have standardized a method for live imaging of post-meiotic histone to protamine switch in intact pupal testes and dissected pupal germ line cysts [18]. Karabasheva, G. and Smyth J.T. established a method for live imaging of male meiotic cytokinesis in intact pupal testes [19]. Here, we demonstrate both in vitro and ex vivo methods to image male meiotic cytokinesis in adult testes. The in vitro method involves isolation of male germline cysts and imaging in a cover glass bottom dish. This in vitro method is of particular interest when the experimental settings require direct treatment of spermatocytes with external agents such as proteins, lipids, and pharmacological agents. Using this method, we show that treatment of spermatocytes with Dynasore, a dynamin inhibitor known to block clathrin-mediated endocytosis [23], also blocks male meiotic cytokinesis by preventing normal assembly of central spindle. The ex vivo method presented here involves dissection of adult testes and imaging on gas permeable membrane dish chambers. Both in vitro and ex vivo methods demonstrated here are extremely valuable to study male meiotic cytokinesis under various treatment conditions and genetic backgrounds.