The endocytic activity of the flagellar pocket in <i>Trypanosoma brucei</i> is regulated by an adjacent phosphatidylinositol phosphate kinase

Demmel, Lars; Schmidt, Katy; Lucast, Louise; Havlicek, Katharina; Zankel, Armin; Koestler, Tina; Reithofer,; P, de Camilli; Warren, Graham

doi:10.1242/jcs.146894

Cited by 38 publications

(50 citation statements)

References 125 publications

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“…These results suggest that the primary site of PI(3,5)P 2 synthesis is on late endosomal, and to a lessor extent lysosomal, membranes, but that the end product resides mostly in the lysosome. This is similar to the situation with PI(4,5)P 2 , where the kinase (TbPIPKA) localizes anterior to the mouth region of the flagellar pocket, but the product localizes quite distinctly to the flagellar pocket proper [56]. …”

Section: Discussionsupporting

confidence: 65%

“…Uptake of endocytic cargo including tomato lectin and transferrin (not shown) was unaffected. This contrasts with depletion of two other PIP's, PI3P (TbVps34 knockdown) and PI(4,5)P 2 (TbPIPKA knockdown), both of which result in defective endocytosis [56, 57], and further supports the specific assignment of TbFab1 activity to PI(3,5)P 2 . In addition, lysosomal delivery, measured as decline in FITC fluorescence as cargo moved to more acidic compartments, was unimpaired by TbFab1 depletion.…”

Section: Discussionsupporting

confidence: 53%

“…These esults agree with similar measurements in yeast and in animals cells in which PI(3,5)P 2 is present at as little as 1% the level of PI(4,5)P 2 [37, 43-45]. A previous study also identified PI(4,5)P 2 as the major PIP 2 species in trypanosomes using methodology that may not have had the sensitivity required to detect low levels of PI(3,5)P 2 [56]. Consequently, this is the first formal demonstration of PI(3,5)P 2 in trypanosomes.…”

Section: Discussionmentioning

confidence: 99%

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The role of the PI(3,5)P2 kinase TbFab1 in endo/lysosomal trafficking in Trypanosoma brucei

Gilden

Umaer

Kruzel

et al. 2017

Molecular and Biochemical Parasitology

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Protein trafficking through endo/lysosomal compartments is critically important to the biology of the protozoan parasite Trypanosoma brucei, but the routes material may take to the lysosome, as well as the molecular factors regulating those routes, remain incompletely understood. Phosphoinositides are signaling phospholipids that regulate many trafficking events by recruiting specific effector proteins to discrete membrane subdomains. In this study, we investigate the role of one phosphoinositide, PI(3,5)P2 in T. brucei. We find a low steady state level of PI(3,5)P2 in bloodstream form parasites comparable to that of other organisms. RNAi knockdown of the putative PI(3)P-5 kinase TbFab1 decreases the PI(3,5)P2 pool leading to rapid cell death. TbFab1 and PI(3,5)P2 both localize strongly to late endo/lysosomes. While most trafficking functions were intact in TbFab1 deficient cells, including both endocytic and biosynthetic trafficking to the lysosome, lysosomal turnover of an endogenous ubiquitinylated membrane protein, ISG65, was completely blocked suggesting that TbFab1 plays a role in the ESCRT-mediated late endosomal/multivesicular body degradative pathways. Knockdown of a second component of PI(3,5)P2 metabolism, the PI(3,5)P2 phosphatase TbFig4, also resulted in delayed turnover of ISG65. Together, these results demonstrate an essential role for PI(3,5)P2 in the turnover of ubiquitinylated membrane proteins and in trypanosome endomembrane biology.

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Section: Discussionsupporting

confidence: 65%

Section: Discussionsupporting

confidence: 53%

Section: Discussionmentioning

confidence: 99%

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The role of the PI(3,5)P2 kinase TbFab1 in endo/lysosomal trafficking in Trypanosoma brucei

Gilden

Umaer

Kruzel

et al. 2017

Molecular and Biochemical Parasitology

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“…For instance, it has recently been shown that a phosphatidylinositol 4-phosphate 5-kinase, TbPIPKA, is present in the FPN (53). This kinase manufactures the phosphatidylinositol 4,5-bisphosphate that is essential for endocytosis.…”

Section: Discussionmentioning

confidence: 99%

A MORN Repeat Protein Facilitates Protein Entry into the Flagellar Pocket of Trypanosoma brucei

Morriswood

Schmidt

2015

Eukaryot Cell

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The parasite Trypanosoma brucei lives in the bloodstream of infected mammalian hosts, fully exposed to the adaptive immune system. It relies on a very high rate of endocytosis to clear bound antibodies from its cell surface. All endo-and exocytosis occurs at a single site on its plasma membrane, an intracellular invagination termed the flagellar pocket. Coiled around the neck of the flagellar pocket is a multiprotein complex containing the repeat motif protein T. brucei MORN1 (TbMORN1). In this study, the phenotypic effects of TbMORN1 depletion in the mammalian-infective form of T. brucei were analyzed. Depletion of TbMORN1 resulted in a rapid enlargement of the flagellar pocket. Dextran, a polysaccharide marker for fluid phase endocytosis, accumulated inside the enlarged flagellar pocket. Unexpectedly, however, the proteins concanavalin A and bovine serum albumin did not do so, and concanavalin A was instead found to concentrate outside it. This suggests that TbMORN1 may have a role in facilitating the entry of proteins into the flagellar pocket.T rypanosoma brucei is an important parasite of humans and domestic animals in sub-Saharan Africa, as the causative agent of sleeping sickness and nagana, respectively. Its complex life cycle involves transitions between tsetse fly vectors (its definitive hosts) and mammalian intermediate hosts. This life cycle involves a number of different cell stages, of which the procyclic form (found in the tsetse fly) and the slender bloodstream form (BSF) (found in the mammalian bloodstream) are the best studied in a laboratory setting. The procyclic form and the BSF of T. brucei share similar cytoskeletal architectures (1, 2).The principal feature of this cytoskeleton is a corset of microtubules that lie directly underneath the plasma membrane and impart to the cell its distinctive shape (3). A single invagination of the plasma membrane, termed the flagellar pocket (FP), constitutes a distinct subdomain and is found at the posterior end of the cell (4). The FP is the site of all endo-and exocytic traffic (5, 6). Abutting the FP membrane is a basal body that nucleates the single flagellum of the trypanosome cell. The flagellum exits the FP and is adhered longitudinally to the cell body along a left-handed helical path (7). Once outside the FP, the axoneme of the flagellum is paralleled by an associated intraflagellar structure called the paraflagellar rod (PFR). The PFR is composed of a paracrystalline lattice and is associated with cellular motility (8). Nucleated adjacent to the basal body is a specialized microtubule quartet that traces around the FP and then underlies the flagellum as far as the anterior end of the cell (4).The small cylinder of membrane that connects the FP to the rest of the plasma membrane constitutes a third subdomain and is called the flagellar pocket neck (FPN) (4). A number of discrete cytoskeletal structures cluster around the FPN membrane on its cytoplasmic face. Of these, the best characterized is an electrondense horseshoe-shaped structure named th...

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“…Xu et al showed that the 5-phosphatase function of INPP5E is counteracted by the PI4P 5-kinase PIPKIγ and that enrichment of PI(4,5)P 2 at the base of the cilium is critical for ciliogenesis [71**]. A PI(4,5)P 2 -enriched zone at the base of the cilium has also been found in T. brucei and C. elegans [53**,72]. Since PI(4,5)P 2 effector proteins include the exocyst, septins, tubby family proteins, and clathrin adaptor proteins [73-76], a PI(4,5)P 2 -enriched domain could mark the ciliary pocket as a specialized site for vesicle exocytosis and endocytosis (Figure 3).…”

Section: Generating a Unique Ciliary Lipid Contentmentioning

confidence: 99%

Permeability barriers for generating a unique ciliary protein and lipid composition

Verhey

Yang

2016

Current Opinion in Cell Biology

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Cilia (and flagella) are microtubule-based protrusions that are found in single or multiple copies on the surface of most eukaryotic cells. Defects in cilia formation and/or function have now been correlated with an expanding spectrum of human genetic diseases termed ciliopathies. Recent work indicates that cilia are indeed a bona fide organelle with a unique protein and lipid content that enables specific cellular functions. Despite the physiological and clinical relevance of cilia, our understanding of how a unique protein and lipid composition is generated for this organelle remains poor. Here we review recent work on the mechanisms that determine the protein and lipid content, and thus the functional outputs, of this unique organelle.

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The endocytic activity of the flagellar pocket in Trypanosoma brucei is regulated by an adjacent phosphatidylinositol phosphate kinase

Cited by 38 publications

References 125 publications

The role of the PI(3,5)P2 kinase TbFab1 in endo/lysosomal trafficking in Trypanosoma brucei

The role of the PI(3,5)P2 kinase TbFab1 in endo/lysosomal trafficking in Trypanosoma brucei

A MORN Repeat Protein Facilitates Protein Entry into the Flagellar Pocket of Trypanosoma brucei

Permeability barriers for generating a unique ciliary protein and lipid composition

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