The membrane was washed three times with PBS and then developed using the FluorChem HD2 system (ProteinSimple Inc.). Immunofluorescence microscopy cells were collected by centrifugation, Cichoric Acid washed once with PBS, adhered to glass coverslips, and fixed in cold methanol (?20 C) for 20 min. aged flagellum attachment zones and are required for cytokinesis initiation. Knockdown of FPRC or CIF4 disrupted the localization of CIF1, suggesting that they function upstream of CIF1. Moreover, depletion of CIF4 abolished FPRC localization, indicating that CIF4 functions upstream of FPRC. Together, these results identify two new cytokinesis regulators in and integrate them into the CIF1-mediated cytokinesis regulatory pathway. These findings highlight the presence of a cytokinesis pathway in that is different from that of its mammalian host and therefore suggest that cytokinesis in could potentially be exploited as a new drug target. belongs to the Excavata supergroup of eukaryotes (1) and is a flagellated unicellular protozoan parasite causing sleeping sickness in humans and nagana in cattle in sub-Saharan Africa. has a complex life cycle, alternating between the insect vector, tsetse travel, and mammalian hosts, and has devastating effects on agriculture and human health in Africa. In the host, the parasite proliferates through binary cell fission along the longitudinal axis of the cell without the involvement of an actomyosin contractile ring, an evolutionarily conserved cytokinetic apparatus in eukaryotes (2). During early stages of the trypanosome cell division cycle, the parasite assembles a new flagellum, which, like the Cichoric Acid aged flagellum, is usually adhered to the cell body via a cytoskeletal structure called the flagellum attachment zone (FAZ)2 (3). The length of the newly assembled flagellum and its associated FAZ determines the cell division plane (4, 5). This is strikingly different from other eukaryotic organisms, in which the cell division plane is usually defined by the position of the central spindle (6), highlighting the unusual mechanism of cytokinesis in which diverged from your eukaryotic ancestor earlier than yeast. Prior to cleavage furrow ingression in remains largely elusive. Nothing is known about how membrane invagination is usually regulated to form the division fold and how cleavage furrow ingression is usually controlled in the absence of the actomyosin contractile ring. The signaling pathway that controls cytokinesis initiation is usually, however, gradually being elucidated. Two evolutionarily conserved protein kinases, the Aurora B kinase homolog TbAUK1 and the Polo-like kinase homolog TbPLK, were first characterized as essential cytokinesis regulators (9,C12), and the two protein kinases appear to function sequentially at the distal tip of the new FAZ (13). Subsequent works recognized a cohort of trypanosome-specific cytokinesis regulators, including three cytokinesis initiation factor (CIF) proteins CIF1, CIF2, and CIF3 (14,C16); two cleavage furrowClocalizing proteins, KLIF and FRW1 (17,C19); a kinetoplastid-specific protein phosphatase called KPP1 (20); and a hook complexClocalizing protein, BOH1, that recruits TbPLK to the new FAZ tip (21). IGFBP3 Through genetic analyses, Cichoric Acid the order of action of these cytokinesis regulators has been decided, with BOH1 functioning at the most upstream of the cytokinesis pathway, followed by TbPLK, the three CIF proteins, TbAUK1, and finally FRW1 and KLIF, acting sequentially at the new FAZ tip and the cleavage furrow (14,C16, 18, 21). Despite these advance, however, much remains to be learned about the functional interplay among these cytokinesis regulators and their mechanistic functions in controlling cytokinesis initiation and/or completion. We aim to delineate the cytokinesis signaling pathway and dissect the mechanisms underlying the unusual mode of cytokinesis in (15, 18). Among these trypanosome-specific proteins is usually a CIF1-interacting protein called FAZ tipClocalizing protein required for Cichoric Acid cytokinesis (FPRC) (18) whose subcellular localization and physiological function have not been characterized in the previous work. FPRC contains two coiled-coil motifs (Fig. 1in trypanosomes (18), we examined their colocalization during the cell cycle by immunofluorescence microscopy. During G1 phase, CIF1 was not detectable, and FPRC was localized at the FAZ tip (Fig. 1= 5 m. = 5 m. indicate the GST and GST fusion proteins. indicate the GST and GST fusion proteins. CIF1 contains a coiled-coil (CC) motif in its N-terminal portion and two Cys-Cys-His-Cys (CCHC)-type zinc finger (ZnF) motifs at the C terminus (Fig. 1and and and show S.D. from three impartial experiments (= 3). = 5 m. = 3). = 5 m. FPRC functions upstream of CIF1 in the cytokinesis regulatory pathway The fact that.