Annelise Sahin

Metabolic status rather than cell cycle signals control quiescence entry and exit

The use of new candidate markers for yeast quiescence reveals that quiescence entry and exit primarily rely on cellular metabolic status and can be uncoupled from the cell cycle.

The Leishmania ARL-1 and Golgi Traffic

We present here the characterisation of the Leishmania small G protein ADP-Ribosylation Factor-Like protein 1 (ARL-1). The ARL-1 gene is present in one copy per haploid genome and conserved among trypanosomatids. It encodes a protein of 20 kDa, which is equally expressed in the insect promastigote and mammalian amastigote forms of the parasite. ARL-1 localises to the Trans-Golgi Network (TGN); N-terminal myristoylation is essential for TGN localisation. In vivo expression of the LdARL-1/Q74L and LdARL-1/T51N mutants (GTP- and GDP-bound blocked forms respectively) shows that GDP/GTP cycling occurs entirely within the TGN. This is contrary to previous reports in yeast and mammals, where the mutant empty form devoid of nucleotide has been considered as the GDP-blocked form. The dominant-negative empty form mutant LdARL-1/T34N inhibits endocytosis and intracellular trafficking from the TGN to the Lysosome/Multivesicular Tubule and to the acidocalcisomes; these defects are probably related to a mislocalisation of the GRIP domain-containing vesicle tethering factors which cannot be recruited to the TGN by the cytoplasmic LdARL-1/T34N. Thus, besides the functional characterization of a new mutant and a better understanding of ARL-1 GDP/GTP cycling, this work shows that Leishmania ARL-1 is a key component of an essential pathway worth future study.

Polarized Growth in the Absence of F-Actin in Saccharomyces cerevisiae Exiting Quiescence

Background Polarity establishment and maintenance are crucial for morphogenesis and development. In budding yeast, these two intricate processes involve the superposition of regulatory loops between polarity landmarks, RHO GTPases, actin-mediated vesicles transport and endocytosis. Deciphering the chronology and the significance of each molecular step of polarized growth is therefore very challenging. Principal Findings We have taken advantage of the fact that yeast quiescent cells display actin bodies, a non polarized actin structure, to evaluate the role of F-actin in bud emergence. Here we show that upon exit from quiescence, actin cables are not required for the first steps of polarized growth. We further show that polarized growth can occur in the absence of actin patch-mediated endocytosis. We finally establish, using latrunculin-A, that the first steps of polarized growth do not require any F-actin containing structures. Yet, these structures are required for the formation of a bona fide daughter cell and cell cycle completion. We propose that upon exit from quiescence in the absence of F-actin, secretory vesicles randomly reach the plasma membrane but preferentially dock and fuse where polarity cues are localized, this being sufficient to trigger polarized growth.

A MAP6-Related Protein Is Present in Protozoa and Is Involved in Flagellum Motility

In vertebrates the microtubule-associated proteins MAP6 and MAP6d1 stabilize cold-resistant microtubules. Cilia and flagella have cold-stable microtubules but MAP6 proteins have not been identified in these organelles. Here, we describe TbSAXO as the first MAP6-related protein to be identified in a protozoan, Trypanosoma brucei. Using a heterologous expression system, we show that TbSAXO is a microtubule stabilizing protein. Furthermore we identify the domains of the protein responsible for microtubule binding and stabilizing and show that they share homologies with the microtubule-stabilizing Mn domains of the MAP6 proteins. We demonstrate, in the flagellated parasite, that TbSAXO is an axonemal protein that plays a role in flagellum motility. Lastly we provide evidence that TbSAXO belongs to a group of MAP6-related proteins (SAXO proteins) present only in ciliated or flagellated organisms ranging from protozoa to mammals. We discuss the potential roles of the SAXO proteins in cilia and flagella function.

BILBO1 Is a Scaffold Protein of the Flagellar Pocket Collar in the Pathogen Trypanosoma brucei

The flagellar pocket (FP) of the pathogen Trypanosoma brucei is an important single copy structure that is formed by the invagination of the pellicular membrane. It is the unique site of endo- and exocytosis and is required for parasite pathogenicity. The FP consists of distinct structural sub-domains with the least explored being the annulus/horseshoe shaped flagellar pocket collar (FPC). To date the only known component of the FPC is the protein BILBO1, a cytoskeleton protein that has a N-terminus that contains an ubiquitin-like fold, two EF-hand domains, plus a large C-terminal coiled-coil domain. BILBO1 has been shown to bind calcium, but in this work we demonstrate that mutating either or both calcium-binding domains prevents calcium binding. The expression of deletion or mutated forms of BILBO1 in trypanosomes and mammalian cells demonstrate that the coiled-coil domain is necessary and sufficient for the formation of BILBO1 polymers. This is supported by Yeast two-hybrid analysis. Expression of full-length BILBO1 in mammalian cells induces the formation of linear polymers with comma and globular shaped termini, whereas mutation of the canonical calcium-binding domain resulted in the formation of helical polymers and mutation in both EF-hand domains prevented the formation of linear polymers. We also demonstrate that in T. brucei the coiled-coil domain is able to target BILBO1 to the FPC and to form polymers whilst the EF-hand domains influence polymers shape. This data indicates that BILBO1 has intrinsic polymer forming properties and that binding calcium can modulate the form of these polymers. We discuss whether these properties can influence the formation of the FPC.

LdFlabarin, a New BAR Domain Membrane Protein of Leishmania Flagellum

During the Leishmania life cycle, the flagellum undergoes successive assembly and disassembly of hundreds of proteins. Understanding these processes necessitates the study of individual components. Here, we investigated LdFlabarin, an uncharacterized L. donovani flagellar protein. The gene is conserved within the Leishmania genus and orthologous genes only exist in the Trypanosoma genus. LdFlabarin associates with the flagellar plasma membrane, extending from the base to the tip of the flagellum as a helicoidal structure. Site-directed mutagenesis, deletions and chimera constructs showed that LdFlabarin flagellar addressing necessitates three determinants: an N-terminal potential acylation site and a central BAR domain for membrane targeting and the C-terminal domain for flagellar specificity. In vitro, the protein spontaneously associates with liposomes, triggering tubule formation, which suggests a structural/morphogenetic function. LdFlabarin is the first characterized Leishmania BAR domain protein, and the first flagellum-specific BAR domain protein.

FPC4: a new cytoskeletal component in T.brucei

Trypanosoma brucei is the causative agent of African sleeping sickness. It possesses a single flagellum, which additional to its mobility role, is an important sensory and signaling organelle [1,2]. The flagellum exits the cell from a membrane invagination, called the flagellar pocket (FP), region where endo- and exocytosis occur. A ring-shaped structure, called the flagellar pocket collar (FPC), encloses the FP defining the flagellum exit point. BILBO1 is the first FPC protein identified and is essential for the FPC and FP biogenesis [2]. A T. brucei gDNA yeast two-hybrid (Y2H) screen identified several BILBO1 protein partners, including FPC4 a protein we are characterizing at the molecular and functional level. Our anti-FPC4 antibody and endogenous FPC4-myc cell confirmed that FPC4 localizes both on, and close to the FPC. RNAi down-regulation of FPC4 has no impact on cell proliferation. However, over-expression of FPC4 leads to filament formation and to a mild, but reduced growth defect. Co-immunolocalization demonstrated that BILBO1 relocalizes to the FP4 filaments indicating BILBO1-FPC4 interaction and the role of FPC4 in FPC structure. Y2H shows that BILBO1 interacts with the C-terminal domain of FPC4 and we are currently identifying the minimal interaction domains, with the long-term objective of a drug screen to block this interaction. Expression of FPC4 in a heterologous system (human cells) suggests that the N-terminal domain binds to microtubules (MT). This was confirmed by in vitro MT binding assays and is ongoing. We are also characterizing FPC4 function in bloodstream form. Our preliminary data suggest that FPC4 could be a FPC-microtubule linker involved in FPC segregation during the cell cycle.

TbSAXO is a MAP6-related protein involved in motility of Trypanosoma brucei flagellum

The microtubules (MTs) of most vertebrate tissue cells will disassemble at low temperature, but some remain cold-stable or resistant to drugs such as nocodazole. It has been shown that MT cold- and nocodazole-resistance is largely due to the association with the class of Microtubule Associated Proteins (MAP) known as MAP6 (previously named STOP for Stable Tubule Only Polypeptide) [1]. MAP6 proteins are expressed only in vertebrates, and have been localized in neurons, astrocytes, oligodendrocytes, fibroblasts, and several tissues. In eukaryotes, the MT-based organelles centrioles, cilia and flagella MT have cold-resistant MTs, but, so far, MAP6 proteins have not been characterized in these organelles. We have recently identified TbSAXO (for Stop AXOneme), a novel flagellar protein in the protozoan parasite Trypanosoma brucei. We show here that TbSAXO is a microtubule stabilizing protein with properties similar, upon cold and nocodazole treatment, to those of the microtubule-stabilizing Mn domains of the MAP6 proteins, thus identifying the first MAP6-related protein in a protozoan. Further, we demonstrate, in the parasite, that TbSAXO is an axoneme-associated protein, which plays a role in flagellum motility. We also show that TbSAXO is the first member of a group of MAP6-related proteins (that we named SAXO proteins) present only in organisms with centrioles / cilia / flagella and ranging from protozoa to mammals, suggesting potential roles of the SAXO proteins in cilia and flagella function. http://mcmp.aquitaine.cnrs.fr/mfp/team_bct_en.php