In addition to Rabs several proteins have been shown

In addition to Rabs, several proteins have been shown to interact with Rabphilin3a, for instance the GluN2A subunit of the NMDA receptor, PSD-95 ([56,57] and references therein), Rabaptin-5, α-actinin [16] and SNAP-25 [21,23,41,61]. Only the latter has been reported to participate in the AR. Exploring whether Rabphilin3a exhibits an additional, later role during the AR, due to the interaction of its C2 domains with SNAP25 and/or membrane phospholipids, is beyond the scope of this manuscript and remains an attractive possibility to be examined in the future. Why would a preformed granule — that has undergone trafficking steps during acrosomal biogenesis and maturation in precursors of the sperm cell in the testis — need two secretory Rabs networked to one another for release is a fascinating question that invites a number of speculative answers. One of them is that a sophisticated link between Rab27 and Rab3 insures the success of fertilization, which depends on the AR taking place in the vicinity of the egg inside the Fallopian tube at the right time. Of the millions of sperm ejaculated into the female tract, a single one fertilizes each oocyte. Furthermore, only a subpopulation of cells challenged with inducers undergoes the AR in vitro even though all sperm in the test tube are equally exposed to them. Whether the cells that exocytose are those where fusion networks like the one elucidated here are in place is not known at this time. The requirement of two secretory Rabs to carry out secretion is not an oddity of the sperm model. Simultaneous presence of Rabs3 and 27 on secretory vesicles and their requirement in regulated exocytosis has been reported in several tissues, for instance purified synaptic vesicles isolated from rat Sildenafil mesylate australia homogenates [52], Weibel–Palade bodies from endothelial cells [3,69], PC12 cells [34,62], pancreatic β-cells [13,47,66], lacrimal acini [46] and human sperm [9,11,43]. It was in the latter cells that the sequence Rab27 → Rab3GEF → Rab3, which gave origin to the discoveries reported in this manuscript, was proposed for the first time.
Conclusions Our findings, generated through in vivo, in vitro and in silico approaches, indicate that Rab27-GTP binds Rabphilin3a, which in turn recruits GRAB, a Rab3GEF that catalyzes the exchange of GDP for GTP on Rab3 in the acrosomal region of human sperm. The contribution of this paper to the sperm biology field is to have generated direct evidence for the role of each and every component of the signaling module Rab27-GTP→Rabphilin3a→GRAB→Rab3-GDP→Rab3-GTP in the AR. The contribution of this paper to the exocytosis field is to provide a detailed characterization of the molecular mechanism through which Rab27 and Rab3 cooperate to achieve dense core granule exocytosis. The following are the supplementary data related to this article.
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Introduction Gene therapy has given hope to the humankind against many deadly genetic diseases and offers the prospect of alterations in the genetic material of an individual for treating a particular condition [[1], [2], [3]]. One of the bottlenecks in this process is efficient delivery of the gene of interest to the target tissue [4]. While a wide array of methods of transporting exogenous nucleic acids in vivo have been developed, there are issues related to both efficiency and toxicity. Viral methods of gene transfer are more efficient but are fraught with safety concerns [5]. To circumvent this, a number of non-viral techniques ranging from mechanical methods to chemically mediated processes are being developed for delivering therapeutic nucleic acids [6]. The use of nanotechnology-based methods in delivering drugs as well as nucleic acids is now a well-recognized area of research and also an evolving discipline [3]. Nanocarriers by virtue of their size, capacity for surface modification and easy synthesis have become useful materials for cellular and tissue delivery of nucleic acids [7]. Nanocarriers can be of diverse nature- ranging from inorganic metal as well as oxide nanoparticles to organic nanocomplexes containing polymers or peptides, liposomes and other nanostructures. Cationic Lipids and cationic polymers have been well explored for nucleic acid delivery [4,8]. Of late, peptide-based gene delivery methods have been found to hold a lot of promise because of their ease of synthesis, biodegradability, low toxicity and a combination of properties like efficient DNA condensation, pH sensitive membrane disruption, efficient targeting and translocation across plasma membrane [3,[9], [10], [11], [12]]. Despite the development of so many approaches and technological advancements, efficiency of nucleic acid delivery is very low and challenging in physiological conditions. Even at a cellular level, the major challenges identified in efficient nucleic acid delivery are uptake of the nucleic acid- vector complex, endosomal escape, cytoplasmic stability and translocation to the nucleus [13]. New methods have been developed for overcoming these challenges, either by developing new vectors or chemical modifications and intelligent strategies [[14], [15], [16]]. In this context, mechanisms of cellular uptake and trafficking of nanoparticles and nanocomplexes have been studied extensively in the literature [17,18]. However the phenomenon of retention and excretion of these materials from cells and the underlying mechanisms involved are explored to a much lesser extent. Cellular retention can be a critical factor influencing gene delivery, as the removal of uptaken vector-nucleic acid complex from the cell will ultimately affect the amount of DNA reaching the nucleus [19]. Therefore it will be interesting to understand the mechanisms involved in the egress of nanoparticles and nanocomplexes from the cell [20]. In addition, many of these nanoparticles are used for other applications like intracellular imaging and delivery of other therapeutics in a targeted manner. Insights into the mechanisms of the entry and exit of nanoparticles from cells can help in honing these information for better design of multifunctional vectors.