David Perez-Morga

INPP5E mutations cause primary cilium signaling defects, ciliary instability and ciliopathies in human and mouse.

The primary cilium is an antenna-like structure that protrudes from the cell surface of quiescent/differentiated cells and participates in extracellular signal processing. Here, we report that mice deficient for the lipid 5-phosphatase Inpp5e develop a multiorgan disorder associated with structural defects of the primary cilium. In ciliated mouse embryonic fibroblasts, Inpp5e is concentrated in the axoneme of the primary cilium. Inpp5e inactivation did not impair ciliary assembly but altered the stability of pre-established cilia after serum addition. Blocking phosphoinositide 3-kinase (PI3K) activity or ciliary platelet-derived growth factor receptor α (PDGFRα) restored ciliary stability. In human INPP5E, we identified a mutation affecting INPP5E ciliary localization and cilium stability in a family with MORM syndrome, a condition related to Bardet-Biedl syndrome. Together, our results show that INPP5E plays an essential role in the primary cilium by controlling ciliary growth factor and PI3K signaling and stability, and highlight the consequences of INPP5E dysfunction.

The trypanolytic factor of human serum.

African trypanosomes (the prototype of which is Trypanosoma brucei brucei) are protozoan parasites that infect a wide range of mammals. Human blood, unlike the blood of other mammals, has efficient trypanolytic activity, and this needs to be counteracted by these parasites. Resistance to this activity has arisen in two subspecies of Trypanosoma brucei — Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense — allowing these parasites to infect humans, and this results in sleeping sickness in East Africa and West Africa, respectively. Study of the mechanism by which T. b. rhodesiense escapes lysis by human serum led to the identification of an ionic-pore-forming apolipoprotein — known as apolipoprotein L1 — that is associated with high-density-lipoprotein particles in human blood. In this Opinion article, we argue that apolipoprotein L1 is the factor that is responsible for the trypanolytic activity of human serum.

A differential role for actin during the life cycle of Trypanosoma brucei

Actin is expressed at similar levels but in different locations in bloodstream and procyclic forms of Trypanosoma brucei. In bloodstream forms actin colocalizes with the highly polarized endocytic pathway, whereas in procyclic forms it is distributed throughout the cell. RNA interference demonstrated that in bloodstream forms, actin is an essential protein. Depletion of actin resulted in a rapid arrest of cell division, termination of vesicular traffic from the flagellar pocket membrane leading to gross enlargement of the pocket, loss of endocytic activity and eventually cell death. These results indicate that actin is required for the formation of coated vesicles from the flagellar pocket membrane, which is the first step in the endocytic pathway. Although loss of actin in procyclic cells did not affect growth, the trans region of the Golgi became distorted and enlarged and appeared to give rise to a heterogeneous population of vesicles. However, the flagellar pocket was not affected. These findings suggest that trypanosomes have different functional requirements for actin during the bloodstream and procyclic phases of the life cycle.

Trypanosoma brucei TBRGG1, a mitochondrial oligo(U)-binding protein that co-localizes with an in vitro RNA editing activity.

We report the characterization of aTrypanosoma brucei 75-kDa protein of the RGG (Arg-Gly-Gly) type, termed TBRGG1. Dicistronic and monocistronic transcripts of theTBRGG1 gene were produced by both alternative splicing and polyadenylation. TBRGG1 was found in two or three forms that differ in their electrophoretic mobility on SDS-polyacrylamide gel electrophoresis gels, one of which was more abundant in the procyclic form of the parasite. TBRGG1 was localized to the mitochondrion and appeared to be more abundant in bloodstream intermediate and stumpy forms in which the mitochondrion reactivates and during the procyclic stage, which possesses a fully functional mitochondrion. This protein was characterized to display oligo(U) binding characteristics and was found to co-localize with an in vitro RNA editing activity in a sedimentation analysis. TBRGG1 most likely corresponds to the 83-kDa oligo(U)-binding protein previously identified by UV cross-linking of guide RNA to mitochondrial lysates (Leegwater, P., Speijer, D., and Benne, R. (1995) Eur. J. Biochem.227, 780–786).

Mechanism of Trypanosoma brucei gambiense resistance to human serum

The African parasite Trypanosoma brucei gambiense accounts for 97% of human sleeping sickness cases. T. b. gambiense resists the specific human innate immunity acting against several other tsetse-fly-transmitted trypanosome species such as T. b. brucei, the causative agent of nagana disease in cattle. Human immunity to some African trypanosomes is due to two serum complexes designated trypanolytic factors (TLF-1 and -2), which both contain haptoglobin-related protein (HPR) and apolipoprotein LI (APOL1). Whereas HPR association with haemoglobin (Hb) allows TLF-1 binding and uptake via the trypanosome receptor TbHpHbR (ref. 5), TLF-2 enters trypanosomes independently of TbHpHbR (refs 4, 5). APOL1 kills trypanosomes after insertion into endosomal/lysosomal membranes. Here we report that T. b. gambiense resists TLFs via a hydrophobic β-sheet of the T. b. gambiense-specific glycoprotein (TgsGP), which prevents APOL1 toxicity and induces stiffening of membranes upon interaction with lipids. Two additional features contribute to resistance to TLFs: reduction of sensitivity to APOL1 requiring cysteine protease activity, and TbHpHbR inactivation due to a L210S substitution. According to such a multifactorial defence mechanism, transgenic expression of T. b. brucei TbHpHbR in T. b. gambiense did not cause parasite lysis in normal human serum. However, these transgenic parasites were killed in hypohaptoglobinaemic serum, after high TLF-1 uptake in the absence of haptoglobin (Hp) that competes for Hb and receptor binding. TbHpHbR inactivation preventing high APOL1 loading in hypohaptoglobinaemic serum may have evolved because of the overlapping endemic area of T. b. gambiense infection and malaria, the main cause of haemolysis-induced hypohaptoglobinaemia in western and central Africa.

Turnover of glycosomes during life-cycle differentiation of Trypanosoma brucei.

Protozoan Kinetoplastida, a group that comprises the pathogenic Trypanosoma brucei, compartmentalize several metabolic systems such as the major part of the glycolytic pathway, in multiple peroxisome-like organelles, designated glycosomes. Trypanosomes have a complicated life cycle, involving two major, distinct stages living in the mammalian bloodstream and several stages inhabiting different body parts of the tsetse fly. Previous studies on non-differentiating trypanosomes have shown that the metabolism and enzymatic contents of glycosomes in bloodstream-form and cultured procyclic cells, representative of the stage living in the insect’s midgut, differ considerably. In this study, the morphology of glycosomes and their position relative to the lysosome were followed, as were the levels of some glycosomal enzymes and markers for other subcellular compartments, during the differentiation from bloodstream-form to procyclic trypanosomes. Our studies revealed a small tendency of glycosomes to associate with the lysosome when a population of long-slender bloodstream forms differentiated into short-stumpy forms which are pre-adapted to live in the fly. The same phenomenon was observed during the short-stumpy to procyclic transformation, but then the process was fast and many more glycosomes were associated with the dramatically enlarged degradation organelle. The observations suggested an efficient glycosome turnover involving autophagy. Changes observed in the levels of marker enzymes are consistent with the notion that, during differentiation, glycosomes with enzymatic contents specific for the old life-cycle stage are degraded and new glycosomes with different contents are synthesized, causing that the metabolic repertoire of trypanosomes is, at each stage, optimally adapted to the environmental conditions encountered.

The attachment of minicircles to kinetoplast DNA networks during replication.

Kinetoplast DNA (kDNA), the trypanosomatid mitochondrial DNA, is a network containing several thousand interlocked minicircles. During kDNA synthesis, minicircles dissociate from the network, and after replication their progeny reattach to the network periphery. Using electron microscopy autoradiography, we found that newly synthesized 3H-labeled minicircles, after short labeling periods, are concentrated in two peripheral zones on opposite sides of the network. These must be minicircle attachment sites, adjacent to the two diametrically opposed complexes of replication proteins observed previously. From the pattern of radiolabeling during longer pulses, we reached the unexpected conclusion that minicircle attachment around the entire network periphery may be due to a relative movement of the kinetoplast and the two complexes. The kinetoplast probably rotates between two fixed complexes.

PI3K Class II α Controls Spatially Restricted Endosomal PtdIns3P and Rab11 Activation to Promote Primary Cilium Function

Summary Multiple phosphatidylinositol (PtdIns) 3-kinases (PI3Ks) can produce PtdIns3P to control endocytic trafficking, but whether enzyme specialization occurs in defined subcellular locations is unclear. Here, we report that PI3K-C2α is enriched in the pericentriolar recycling endocytic compartment (PRE) at the base of the primary cilium, where it regulates production of a specific pool of PtdIns3P. Loss of PI3K-C2α-derived PtdIns3P leads to mislocalization of PRE markers such as TfR and Rab11, reduces Rab11 activation, and blocks accumulation of Rab8 at the primary cilium. These changes in turn cause defects in primary cilium elongation, Smo ciliary translocation, and Sonic Hedgehog (Shh) signaling and ultimately impair embryonic development. Selective reconstitution of PtdIns3P levels in cells lacking PI3K-C2α rescues Rab11 activation, primary cilium length, and Shh pathway induction. Thus, PI3K-C2α regulates the formation of a PtdIns3P pool at the PRE required for Rab11 and Shh pathway activation.

A receptor-like flagellar pocket glycoprotein specific to Trypanosoma brucei gambiense.

Trypanosoma brucei gambiense and T. b. rhodesiense are protozoan parasites causing sleeping sickness in humans due to their resistance to lysis by normal human serum (NHS). Based on the observation that the resistance gene of T. b. rhodesiense encodes a truncated form of the variant specific glycoprotein (VSG), we cloned a similar gene in T. b. gambiense using reverse transcription-linked polymerase chain reaction with VSG-specific primers. This gene, termed TgsGP for T. gambiense-specific glycoprotein, was found to be specific to T. b. gambiense. It is located close to a telomere and is transcribed by a pol II RNA polymerase, only at the bloodstream stage of the parasite development. TgsGP encodes a 47-kDa protein consisting of a N-terminal VSG domain presumably provided with a glycosylphosphatidylinositol (GPI) anchor sequence, similar to the pESAG6 subunit of the trypanosomal transferrin receptor. TgsGP is located in the flagellar pocket, and contains the linear N-linked polyacetyllactosamine characteristic of the endocytotic machinery of T. brucei. These observations strongly suggest that TgsGP is a T. b. gambiense specific receptor. Since stable expression of this protein in T. b. brucei did not confer resistance to NHS, TgsGP may either need another factor to achieve this purpose or fulfils another function linked to adaptation of the parasite to man.

The molecular arms race between African trypanosomes and humans

Humans can survive bloodstream infection by African trypanosomes, owing to the activity of serum complexes that have efficient trypanosome-killing ability. The two trypanosome subspecies that are responsible for human sleeping sickness — Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense — can evade this defence mechanism by expressing distinct resistance proteins. In turn, sequence variation in the gene that encodes the trypanosome-killing component in human serum has enabled populations in western Africa to restore resistance to T. b. rhodesiense, at the expense of the high probability of developing kidney sclerosis. These findings highlight the importance of resistance to trypanosomes in human evolution.