T Lymphocyte-triggering Factor of African Trypanosomes Is Associated with the Flagellar Fraction of the Cytoskeleton and Represents a New Family of Proteins That Are Present in Several Divergent Eukaryotes*

The trypanosome cytoskeleton consists almost entirely of microtubule-based structures. Although α- and β-tubulin from Trypanosoma brucei have been well characterized, much less is known about other cytoskeleton-associated proteins in trypanosomes. Using biochemical fractionation, we demonstrate here that T lymphocyte-triggering factor (TLTF) from T. brucei is a component of the detergent-resistant and Ca2+-resistant fraction of the parasite cytoskeleton. This fraction contains the flagellar apparatus and a subset of cytoskeletal protein complexes that together function in cell motility, cytokinesis, and organelle inheritance. We also show that TLTF-related genes are present in several highly divergent eukaryotic organisms. Although the function of the corresponding proteins is not known, the mammalian TLTF-like gene (GAS11; growtharrest-specific gene 11) is up-regulated in growth-arrested cells and is a candidate tumor suppressor (Whitmore, S. A., Settasatian, C., Crawford, J., Lower, K. M., McCallum, B., Seshadri, R., Cornelisse, C. J., Moerland, E. W., Cleton-Jansen, A. M., Tipping, A. J., Mathew, C. G., Savnio, M., Savoia, A., Verlander, P., Auerbach, A. D., Van Berkel, C., Pronk, J. C., Doggett, N. A., and Callen, D. F. (1998) Genomics 52, 325–331), suggestive of a role in coordinating cytoskeleton activities. Consistent with this possibility, we show that the human GAS11 protein contains a 144-amino acid domain that co-localizes with microtubules when fused to the green fluorescent protein and expressed in mammalian cells. These findings suggest that TLTF represents a newly defined protein family, whose members contribute to cytoskeleton function in species as diverse as protozoa and mammals.

The cytoskeleton is a central and defining feature of all eukaryotic cells. In addition to providing each cell with its unique shape and form, the cytoskeleton plays a central role in cell division, cell motility and subcellular trafficking of proteins and organelles. In most eukaryotic cells, the cytoskeleton consists of three major filament systems, microtubules, intermediate filaments, and actin microfilaments, which form an interwoven scaffolding that permeates the cytoplasmic space of the cell. In contrast, the cytoskeleton of trypanosomes is composed almost entirely of microtubules and microtubule-associated proteins and is further distinguished by the conspicuous absence of transcellular filaments (1,2).
African trypanosomes are protozoan parasites that cause African trypanosomiasis, a fatal disease commonly called African sleeping sickness in humans and nagana in cattle. The two most prominent features of the trypanosome cytoskeleton are the flagellum, including the axoneme and paraflagellar rod, and a longitudinal array of cytoplasmic microtubules, collectively referred to as the subpellicular corset (2; see also Fig. 7). Two additional cytoskeletal structures observed by electron microscopy are an electron-dense filament that defines the cytoplasmic side of the flagellum attachment zone (FAZ) 1 and a specialized group of four reticulum-associated microtubules adjacent to the FAZ filament (2)(3)(4). The flagellum, subpellicular corset, FAZ filament, and special quartet of microtubules all run parallel to the long axis of the cell and together function in organelle inheritance (5,6), cell division (6,7), and cell motility (8).
As is the case in other eukaryotes, trypanosome microtubules are composed of two main structural subunits (␣-and ␤-tubulin) that interact with a number of less abundant microtubule-associated proteins (4,9). Trypanosome ␣and ␤-tubulin have been well characterized and are similar in sequence to those of other eukaryotes (4). They are also subject to posttranslational modifications that are observed in other tubulins, including acetylation, glutamylation, and reversible detyrosination (4,10). Despite these similarities, trypanosome microtubules exhibit a number of unusual features that make them an attractive target for the development of anti-trypanosomal drugs (1,11). These features include a highly ordered and uniformly polarized organization, extensive intermicrotubule and membrane-microtubule cross-links, and high stability at low temperatures (1,6). Trypanosome microtubules also remain intact throughout the cell cycle (12) and are resistant to anti-microtubule agents that are effective on mammalian cells (1,11). Since at least some of these distinguishing features have been attributed to non-tubulin cytoskeleton-associated/ microtubule-associated proteins (1), a number of biochemical and immunological approaches have been taken to identify such proteins in trypanosomes (2,9,13,14). In addition to increasing our understanding of the cytoskeletal architecture of African trypanosomes, the heavy dependence of this early diverging eukaryote on microtubules for cytoskeleton function makes it likely that these efforts will facilitate the identification of novel microtubule-binding proteins (9).
In previous studies (15), we showed that a fusion protein, containing the green fluorescent protein (GFP) fused to T lymphocyte-triggering factor (TLTF) from Trypanosoma brucei, is localized to punctate structures near the trypanosome flagellar pocket, a specialized invagination of the plasma membrane that forms where the flagellum emerges from the cytoplasm (16). Deletion analysis of TLTF identified an internal 144amino acid domain that directs GFP to an electron-dense structure on the cytoplasmic side of the anterior flagellar pocket membrane (17). The analogous segment of a related human protein (GAS11) directs GFP to the extreme posterior end of the trypanosome cell (17), a position that has structural features in common with the flagellar pocket (1,18) and corresponds to the "plus" end of the subpellicular microtubules (6). These results, combined with the targeting properties of TLTF targeting domain mutants, led us to suggest that TLTF interacts with the cytoskeleton (17). In the present work, we use biochemical fractionation to demonstrate that TLTF is associated with the trypanosome cytoskeleton. We also show that TLTF-related genes are present in a wide variety of organisms and that a related human protein (GAS11) contains a novel microtubule binding domain.
Construction of Plasmids for Stable Transfections-All plasmids are based on pHD496 (21). To generate pHD496:GFP, the GFP coding sequence was removed from pHD:HX-GFP (17) as a HindIII/BamHI fragment and inserted into the HindIII/BamHI sites of pHD496. To create pHD496:TLTF, we employed a multistep cloning strategy. The 1362-bp TLTF coding sequence has a unique HindIII site at position 1099. The last 263 bp of the TLTF coding sequence plus 120 bp of 3Ј-untranslated region were excised from plasmid pHD:GFP-CD (17) as a HindIII/BamHI fragment and inserted into the HindIII/BamHI sites of pHD496, yielding plasmid pHD496:TLTF-CTerm. The first 1099 bp of the TLTF open reading frame was introduced into pHD496:TLTF-CTerm as follows. Polymerase chain reaction amplification with primers bearing EcoRI restriction sites was used to place EcoRI sites on both sides of the TLTF open reading frame. This polymerase chain reaction product was cloned into the EcoRI site of pBluescript SK II (Stratagene), and restriction mapping was used to identify a clone in which the TLTF start codon is on the EcoRV side of the polylinker. The 5Ј portion of the TLTF coding sequence was excised from this clone by digestion with HindIII. This HindIII fragment was inserted into the unique HindIII site of pHD496:TLTF-CT and checked for orientation by restriction mapping. The resulting plasmid, pHD496:TLTF, contains the entire TLTF coding sequence followed by 120 bp of the TLTF 3Ј-untranslated region. The sequences of all polymerase chain reactionamplified regions and cloning site junctions were confirmed by automated fluorescent DNA sequencing at the University of Iowa DNA sequencing facility.
RNA Preparation and Northern Blotting-RNA was prepared from trypanosomes with RNeasy reagent (Qiagen) according to the manufacturer's instructions. Total RNA (5 g) was analyzed by Northern blot analysis as described previously (22), using 32 P-labeled DNA containing the entire TLTF open reading frame as a probe. COS-7 Cell Transfections-COS-7 cells (American Type Culture Collection) were maintained at 37°C in 5% CO 2 and were grown to confluence in RPMI medium supplemented with 10% heat-inactivated fetal calf serum. DNA fragments encoding GFP or amino acids 115-258 of GAS11 fused to GFP were excised from plasmids pHD:HX-GFP and pHD:HumanHI-GFP, respectively (17). These fragments were ligated into the HindIII/BamHI sites of the pcDNA3 mammalian expression vector (Invitrogen Corp., Carlsbad, CA). For transfection, COS-7 cells were trypsinized, harvested by centrifugation, washed in PBS-II (PBS containing 0.5 mM MgOAc and 0.1 mM CaCl 2 ), and then resuspended in PBS-II at 1 ϫ 10 7 cells/ml. Aliquots of 0.4 ml were placed in 0.2-cm electroporation cuvettes (Bio-Rad) with 15 g of plasmid DNA and electroporated with one pulse of 300 V, 250 microfarads using a Bio-Rad Gene Pulser. Transfected cells were transferred to fresh medium for 48 h and then processed for immunofluorescence.
Indirect immunofluorescence assays were performed essentially as described (23). Cells were extracted with 0.5% Triton X-100 in a microtubule-stabilizing buffer (24) prior to fixation. The monoclonal antibody E7, directed against ␤-tubulin, was developed by (25) and obtained from the Developmental Studies Hybridoma Bank maintained by the University of Iowa Department of Biological Sciences. E7 tissue culture supernatant was used at a dilution of 1:400 in PBS containing 0.1% heat-inactivated fetal calf serum. Primary antibodies were detected with Cy3-conjugated donkey ␣-mouse IgG (Jackson Laboratories) at a dilution of 1:500. Cells were visualized by laser-scanning confocal microscopy as described previously (17). Omission of primary or secondary antibodies resulted in no immunofluorescence (not shown). Tubulin immunofluorescence (red) and GFP fluorescence (green) were captured on separate channels and superimposed using Adobe Photoshop 4.0.1 (Adobe Systems, San Jose, CA).
Protein Extracts, Immunoprecipitation, Subcellular Fractionation, and Western Blotting-Total protein extracts were prepared and analyzed by Western blotting as described previously (17). The ␣-TLTF antibodies are described below. The ␣-BiP (26) and ␣-Hsp70 (cytoplasmic heat shock protein) antibodies (27) were generously provided by J. Bangs (University of Wisconsin, Madison). The E7 ␣-tubulin monoclonal antibody is described above. For immunoprecipitation, procyclic trypanosomes were labeled with 100 Ci/ml 35 S methionine/cysteine (Expre 35 S 35 S; PerkinElmer Life Sciences) for 6 h as described (28). After labeling, a 10-fold excess of unlabeled cysteine and methionine was added, and cells were harvested by centrifugation, washed twice with PBS, resuspended in boiling buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.1 mM EDTA, 0.5% Tween 20), and boiled for 4 min. Lysates were centrifuged for 2 min to remove insoluble debris, and 7 ϫ 10 6 cell equivalents were immunoprecipitated as described previously (29). Immunoprecipitations utilized 25 l of affinity-purified ␣-TLTF-TT1 or 25 l of polyclonal ␣-GFP antibodies (CLONTECH). Preliminary experiments demonstrated that this quantity of ␣-TLTF-TT1 immunoprecipitates all TLTF in the sample (not shown). Immunoprecipitated proteins were separated by SDS-PAGE and visualized by fluorography. Fractionation of hypotonically lysed trypanosomes (Fig. 3) was performed as described (26). Detergent-extracted cytoskeletons and Ca 2ϩ -resistant flagellar fractions (Fig. 4) were obtained according to the method of Robinson and colleagues (30). All fractions were monitored by phasecontrast microscopy to ensure that lysis and detergent/CaCl 2 extraction was complete. Equal cell equivalents of each fraction were resolved by SDS-PAGE and subjected to Western blot analysis. For Western blot analysis, all antibodies were used at a dilution of 1:500 -1:1000.
Production and Affinity Purification of ␣-TLTF Antisera-The ␣-TLTF-TT1 and ␣-TLTF-TT4 rabbit polyclonal antisera have been described previously (15). These antisera were affinity-purified using Immobilon-P polyvinylidene difluoride strips (Millipore Corp.) containing recombinant TLTF-thioredoxin fusion protein (15) and incubated with Immobilon-P polyvinylidene difluoride strips containing recombinant thioredoxin to remove ␣-thioredoxin antibodies. For ␣-TLTF-Pep4, a synthetic peptide, "Pep4" (CAAQTANIASLPRSNFE), containing the C-terminal 14 amino acids of TLTF plus two alanine spacer residues and a cysteine at the N terminus for cross-linking was synthesized by a commercial vendor, Research Genetics, Inc. (Huntsville, AL). 10 mg of this peptide was coupled to keyhole limpet hemocyanin for immunizations or to bovine serum albumin for affinity purification, using standard methods (31). Immunization of rabbits with 500 g of the KLH-Pep4 conjugate was done according to standard methods (31) with boosts at 3, 7, and 13 weeks. Immune sera were collected 2-3 weeks after each boost and tested by enzyme-linked immunosorbent assay (31) for titer against the BSA-Pep4 conjugate. The immune serum with highest titer was confirmed to recognize TLTF on Western blots (not shown) and then affinity-purified using the BSA-Pep4 conjugate coupled to cyanogen bromide-activated Sepharose (Sigma).
Trypanosome Immunofluorescence Assays-Trypanosomes were harvested by centrifugation at 1000 ϫ g and washed once in PBS. Whole cells were settled directly onto chilled poly-L-lysine coated coverslips. Cytoskeletons and flagella were prepared in solution (30), prior to being settled onto chilled poly-L-lysine-coated coverslips. All coverslips were washed with PBS to remove unattached material, fixed as indicated in Table I, and either rehydrated (for methanol/acetone fixations) or quenched (for aldehyde fixation) using standard procedures (31). Blocking agents, primary antibody, and secondary antibody were added as indicated in Table I. After each antibody incubation, coverslips were washed at least three times in PBS or in PBS containing 0.1-0.5% Tween 20 or Triton X-100 and then incubated with ␣-rabbit secondary antibodies conjugated to fluorescein isothiocyanate or Texas Red (Molecular Probes, Inc., Eugene, OR). Coverslips were mounted in Vectashield TM and sealed prior to visualization on an Olympus BH2 phasecontrast microscope equipped with a 100-watt mercury lamp.

TLTF Is a 54-kDa Protein That Is Expressed in Both Insect
Form and Bloodstream Form Trypanosomes-Previous results from in vivo targeting studies suggested that TLTF might interact with the trypanosome cytoskeleton (17). To explore this possibility further, we employed ␣-TLTF antibodies to examine the behavior of TLTF in biochemical fractionation experiments. Polyclonal antibodies (␣-TLTF-TT1), raised against full-length, recombinant TLTF (15), were affinity-purified by adsorption to TLTF on polyvinylidene difluoride membranes (see "Experimental Procedures"). These affinity-purified antibodies recognize two proteins of 41 and 54 kDa in extracts from insect form trypanosomes (Fig. 1A, lane WT). As expected, fusion proteins containing TLTF fused to either the C terminus or N terminus of GFP are recognized by ␣-GFP antisera and by affinity-purified ␣-TLTF-TT1 antibodies (Fig. 1A, lanes GT and TG). Preimmune sera do not recognize the 41-or 54-kDa endogenous trypanosome proteins or the GFP fusion proteins (not shown). Interestingly, expression of the 41-kDa protein is subject to developmental regulation, since it is absent in bloodstream form trypanosomes (Fig. 1A, lane BF).
The TLTF cDNA encodes a 453-amino acid protein with a predicted molecular mass of 54 kDa (15). This size corresponds well with the size of the larger (54-kDa) endogenous trypanosome protein in Fig. 1A, and with the size of the TLTF-GFP fusion proteins (29 ϩ 54 ϭ 83 kDa) in Fig. 1A (asterisk, lanes GT and TG). Since Northern blot analysis reveals that the TLTF gene is expressed as a single 1.8-kilobase mRNA in both insect form and bloodstream form parasites (Fig. 1B), we were surprised to see cross-reactivity to the smaller (41-kDa) protein in Western blots. A 13-kDa difference in protein molecular weight (54 versus 41 kDa) corresponds to an mRNA size difference of approximately 350 nt, which would be readily detected by Northern blot analysis. Hence, the 41-kDa protein is not simply the product of a truncated TLTF mRNA. Rather, this protein is either encoded by a separate gene or is derived from the 54-kDa protein through post-translational processing.
Since trypanosome proteins that are developmentally regulated are usually important for survival of the parasite in a particular host environment, we considered the possibility that the 41-kDa protein might arise through developmental stagespecific processing of the 54-kDa protein. We set out to conduct a pulse-chase experiment to determine whether the 54-kDa protein could be pulse-labeled in vivo and then processed into the 41-kDa protein over time. Unfortunately, despite the fact FIG. 1. TLTF is a 54-kDa protein expressed in both insect form and bloodstream form trypanosomes. A, total protein extracts were prepared from wild type insect form trypanosomes (WT), wild type bloodstream form trypanosomes (BF), or insect form trypanosomes engineered to express GFP-TLTF (GT) or TLTF-GFP (TG) fusion proteins. Proteins were separated by SDS-PAGE, transferred to nitrocellulose membranes, and probed with ␣-GFP polyclonal antisera or affinity-purified ␣-TLTF-TT1 antibodies as indicated. All samples from insect form parasites were run on a single gel, and bloodstream form samples were run on a separate gel. The sizes and positions of prestained protein MW standards are indicated. Black arrowhead, TLTF; open arrowhead, 41-kDa ␣-TLTF-TT1 cross-reactive protein; asterisk, TLTF-GFP fusion proteins. B, Northern blot of total RNA prepared from wild type insect form (IF) and bloodstream form (BF) trypanosomes probed with a 32 P-labeled TLTF cDNA. that affinity-purified ␣-TLTF-TT1 antibodies readily immunoprecipitate the 35 S-labeled 54-kDa protein, they do not immunoprecipitate the 41-kDa protein ( Fig. 2A, lane 1). Western blot analysis of the supernatant from this immunoprecipitation confirms that the 41-kDa protein remains in the supernatant and is not degraded during the experiment (Fig. 2B). The fact that neither the 54-kDa protein nor any rabbit ␣-TLTF-TT1 IgG heavy chain is detected by the anti-rabbit secondary antibody used for this Western blot (Fig. 2B) demonstrates that the immunoprecipitation is quantitative.
Since we were unable to use pulse-chase experiments to determine whether the 41-kDa protein is derived from the 54-kDa protein, we took the alternative approach of generating trypanosome cells that overexpress the TLTF open reading frame. Western blots of protein extracts from these TLTF overexpressors show a dramatic increase in the amount of 54-kDa protein, with no corresponding increase in the abundance of the 41-kDa protein (Fig. 2C). Furthermore, ␣-TLTF antisera raised against either the N-terminal (␣-TLTF-TT4) or C-terminal (␣-TLTF-Pep4) portion of TLTF recognize the 54-kDa protein but not the 41-kDa protein (Fig. 2D). Based on these results, we conclude that the 54-kDa protein is TLTF and that the 41-kDa protein most likely is a protein that is distinct from TLTF but has an epitope that is shared with TLTF. TLTF Is Associated with the Trypanosome Cytoskeleton-When total cell extracts from hypotonically lysed trypanosomes are centrifuged at 1000 ϫ g, the vast majority of cellular proteins, including proteins that serve as markers for the endoplasmic reticulum (BiP; binding protein; Ref. 26) and cytoplasm (Hsp70; heat shock protein 70; Ref. 32) are released into the supernatant (Fig. 3, A-C) as has been reported previously (26). Since trypanosome microtubules and associated proteins are highly resistant to physical and chemical disruption (30), they are expected to sediment under these conditions. Hence, the major protein of about 55 kDa seen in the Coomassiestained P1 sample (Fig. 3A) most likely corresponds to tubulin. Western blots probed with a monoclonal antibody directed against ␤-tubulin confirm that microtubules remain in the 1000 ϫ g pellet (Fig. 3D, lane P1). Contrary to most cellular proteins (Fig. 1, A-C), both TLTF and the 41-kDa protein co-sediment with microtubules in these experiments (Fig. 3E). TLTF behaves somewhat differently than the 41-kDa protein, since TLTF is quantitatively pelleted, whereas a small amount of the 41-kDa protein remains in the supernatant. Although this difference is slight, it was consistently observed in several experiments (not shown). In addition to the microtubule cytoskeleton, the low speed pellet obtained here is expected to contain nuclei and mitochondria (32) as well as large fragments of surface membrane and unlysed cells. The nearly complete release of endoplasmic reticulum (BiP), and cytoplasmic (Hsp70) marker proteins, as well as microscopic examination of the P1 pellet (not shown) demonstrates that cell lysis is essentially complete.
Since the biochemical fractionation of TLTF is consistent with that of a cytoskeletal protein, we tested this possibility directly by isolating intact trypanosome cytoskeletons and examining these preparations for the presence of TLTF. Robinson et al. (30) have exploited the stability and organization of trypanosome microtubules to develop a procedure for isolating intact cytoskeletons. In this procedure, cellular membranes are removed by extraction with nonionic detergents in the presence of a Ca 2ϩ -chelating agent. Under these conditions, the cytoskeleton remains intact and can be isolated by low speed (1000 ϫ g) centrifugation. This isolated cytoskeleton has the same shape and organization observed in intact cells and retains its major structural components (microtubules and the paraflagellar rod) (12) plus a variety of less abundant cytoskeleton-associated/microtubule-associated proteins (4). We find that TLTF remains quantitatively associated with intact, detergent-extracted cytoskeletons (Fig. 4, lane P1). As expected (30), nearly all cellular tubulin remains polymerized and therefore sediments at 1000 ϫ g (Fig. 4, lane P1), demonstrating the resistance of the microtubule-based cytoskeleton to this treatment. Unlike TLTF, the 41-kDa protein is released into the supernatant by detergent extraction (Fig. 4, lane S1), indicating that this protein interacts with the membrane in the hypotonic P1 pellet (Fig. 3E), whereas TLTF interacts with the

cytoskeleton.
TLTF Remains Associated with the Flagellar Fraction of the Cytoskeleton after Depolymerization of Subpellicular Microtubules with Ca 2ϩ -The two most prominent features of the trypanosome cytoskeleton are the flagellar apparatus and the subpellicular microtubule corset (12). The flagellum contains a classical set of "9 ϩ 2" axoneme microtubules attached to a filamentous structure called the paraflagellar rod (33). The subpellicular microtubule corset is a highly ordered, helical array of parallel microtubules that run parallel to the long axis of the cell and are cross-linked to the plasma membrane (pellicle) (4, 6). These subpellicular microtubules can be selectively depolymerized by treating detergent-extracted cytoskeletons with Ca 2ϩ or high salt concentrations (30), leaving the flagellar axoneme, paraflagellar rod, and a subset of cytoplasmic cytoskeletal structures (Ref. 34; see Fig. 7) intact. This detergentresistant and Ca 2ϩ -resistant "flagellar fraction" can then be separated from the solubilized subpellicular cytoskeleton by centrifugation (30).
Upon depolymerization of the subpellicular microtubules, we find that 100% of TLTF remains associated with the flagellar fraction, even after extensive washing with 3 mM CaCl 2 and 1% Nonidet P-40 (Fig. 4, lane P2). The release of 50 -60% of tubulin into the soluble fraction (Fig. 4, lane S2) is consistent with earlier reports (35) and, together with microscopic examination of the flagellar fraction (not shown), demonstrates that Ca 2ϩ extraction was effective at depolymerizing the subpellicular microtubules. These results demonstrate that TLTF's association with the cytoskeleton does not require intact subpellicular microtubules and indicate that TLTF is part of the subset of flagellum-associated cytoskeletal structures that are resistant to detergent and Ca 2ϩ extraction (see below) (2,34).
Subcellular Location of TLTF-We previously showed that a TLTF-GFP fusion protein is localized to small spots within the cell that are concentrated in the vicinity of the flagellar pocket (15). Deletion analysis of TLTF identified an internal 144amino acid targeting domain (amino acids 114 -257) that directs GFP to an electron-dense structure on the cytoplasmic side of the anterior flagellar pocket membrane (17). Mutations in this domain misdirect GFP to the cytoplasm or into the flagellum (17). To follow up on these GFP studies, we conducted immunofluorescence experiments using three different rabbit polyclonal antisera directed against TLTF. Antibodies ␣-TLTF-TT1 and ␣-TLTF-TT4 were raised against thioredoxin fusion proteins containing either full-length TLTF (TT1) or the first 145 amino acids of TLTF (TT4) (15). A third antibody, ␣-TLTF-Pep4, was raised against the C-terminal 14 amino acids of TLTF (see "Experimental Procedures"). Each of these antibodies was affinity-purified (see "Experimental Procedures") and FIG. 3. TLTF is associated with the membrane/cytoskeleton fraction of protein extracts prepared by hypotonic lysis. Total protein extracts from wild type trypanosomes were prepared by hypotonic lysis and subjected to centrifugation at 1000 ϫ g. The crude lysate (L), 1000 ϫ g supernatant (S1), and pellet (P1) were separated by SDS-PAGE, stained with Coomassie Blue (A), or subjected to Western blot analysis (B-E) using antibodies directed against the indicated proteins (B-D), or ␣-TLTF-TT1 (E). The position of TLTF and the 41-kDa protein are indicated with black and open arrowheads, respectively. BiP and Hsp70 are marker proteins for the endoplasmic reticulum (26) and cytoplasm (32), respectively, and serve as controls to demonstrate that cell lysis is essentially complete. The sizes and positions of prestained protein molecular mass standards are indicated.

FIG. 4. TLTF is a component of the flagellar fraction of the trypanosome cytoskeleton.
To isolate intact trypanosome cytoskeletons, whole cell lysates were prepared by extraction with 1% Nonidet P-40 as described under "Experimental Procedures." Intact cytoskeletons (P1) were separated from detergent-soluble proteins (S1) by centrifugation at 1000 ϫ g. These detergent-extracted cytoskeletons (P1) were further incubated with 3 mM CaCl 2 to depolymerize the subpellicular cytoskeleton (30), leaving the flagellar microtubules and associated structures intact (see "Experimental Procedures"). The Ca 2ϩ -resistant flagellar fraction (P2) was separated from Ca 2ϩ -soluble proteins (S2) by centrifugation at 16,000 ϫ g. All samples were separated by SDS-PAGE and subjected to Western blot analysis using antibodies against the indicated proteins. shown to recognize TLTF in total protein extracts from wild type trypanosomes (Figs. 1 and 2), as well as purified, Histagged TLTF (not shown).
Despite the specificity and high titer of these independent preparations of ␣-TLTF antibodies in Western blots, our efforts to use them for immunofluorescence have been unsuccessful. We have conducted an exhaustive and systematic comparison of fixation, permeabilization, and blocking agents as well as a wide range of antibody dilutions, incubation times, and temperatures (Table I). We have also used 0.5% SDS treatment in an effort to expose obscured epitopes and have employed whole cells, cytoskeletons, and isolated flagella as starting material. Primary antibodies against tubulin (25) and CRAM (cysteinerich acidic membrane protein) (36), used as positive controls for immunofluorescence, show the expected staining patterns (not shown). A TLTF-null trypanosome cell line 2 was used as a negative control in these experiments. In all immunofluorescence experiments conducted thus far, none of the three TLTF antibody preparations give a signal above the background seen with TLTF-null trypanosomes. Rather, these antibodies give either no signal or a background signal consisting of spots of fluorescence along the flagellar cytoskeleton, which is also seen with preimmune sera from a variety of nonimmunized rabbits and mice (not shown). The presence of trypanosome cytoskeleton cross-reactive antibodies in naive animals has also been reported by others (37). Based on these results, we suspect that TLTF may be tightly associated with other cytoskeletal proteins and is inaccessible to our antibodies under the immunofluorescence conditions employed. This possibility is supported by our biochemical fractionation data (Figs. 3 and 4) and is consistent with the observation that the TLTF amino acid sequence is predicted to form multiple coiled-coils (17), which mediate protein-protein interactions and are common features of other cytoskeletal proteins (33,38).

TLTF-like Genes Are Present in a Wide
Variety of Organisms-BLAST searches of DNA data bases identified a human EST and a mouse cDNA that encode nearly identical proteins with extensive sequence similarity (60%) to TLTF (15). The mouse gene was isolated in a screen for genes that are upregulated in growth-arrested cells and is designated as Gas11 (growth arrest-specific gene 11). 3 The corresponding human GAS11 gene was recently implicated by Whitmore et al. (40) as a candidate tumor suppressor, since its deletion is commonly associated with breast and prostate cancers. Using the TLTF amino acid sequence to perform BLAST searches, we have now identified partial length genes for TLTF-like proteins in the nucleotide data bases for zebrafish, Trypanosoma cruzi, Schistosoma mansoni, Drosophila melanogaster, and Chlamydomo-nas reinhardtii (Fig. 5). Notably, we have not identified any TLTF sequences in the Saccharomyces cerevisiae genome. Alignment of these partial-length TLTF-like proteins, which correspond to TLTF amino acids 33-247 (Fig. 5A) and 269 -429 (Fig. 5B), reveals several regions of highly conserved amino acid sequence. Aside from a few candidate phosphorylation and glycosylation sites (not shown), these proteins contain no previously identified sequence motifs. They are, however, all predicted to have extensive ␣-helical secondary structures and to form multiple coiled-coils (38,41). Fig.  5A, it is noteworthy that the previously identified flagellar pocket targeting domain of TLTF (amino acids 114 -257) (17) is 70% identical to the corresponding sequence that is known for the T. cruzi protein (amino acids 116 -247). As we reported previously, the last half of this targeting domain corresponds to one of the most conserved regions between TLTF and the human GAS11 protein (17). Our finding that TLTF is a component of trypanosome cytoskeleton suggests that some of the conserved amino acids in GAS11 might correspond to sequences that interact with the cytoskeleton in mammalian cells. This possibility is consistent with our earlier observation that GAS11 amino acids 115-258, which are analogous to the TLTF targeting domain (17), direct a GFP fusion protein to the extreme posterior of the trypanosome cell (17), a position that corresponds to the plus-ends of the trypanosome's cytoplasmic microtubules (6). To determine whether GAS11 amino acids 115-258 interact with mammalian microtubules, we expressed this same fusion protein in mammalian cells and found it to colocalize precisely with microtubules (Fig. 6). This localization pattern is stable to detergent extraction, as expected for a microtubule-binding protein (24), and is disrupted by the microtubule-destabilizing agent, nocodazole (Fig. 6I). The punctate distribution of GAS11-(115-258)-GFP along a subset of microtubules is strikingly similar to that seen for CLIP-170 (24), a "cytoplasmic linker protein" that links subcellular organelles to microtubules and has been implicated in regulating the assembly of microtubules at their plus-end (42)(43)(44). DISCUSSION The trypanosome cytoskeleton is distinguished from that of most eukaryotic cells by the fact that it does not contain any detectable trans-cellular filaments and is composed almost entirely of microtubules and microtubule-associated proteins (4). In contrast to the major microtubule subunits (␣-and ␤-tubulin), which have been well characterized (4), relatively little is known about microtubule-associated/cytoskeleton-associated proteins in trypanosomes. Given the importance of these proteins in contributing to the unique properties of the trypanosome cytoskeleton (1, 2), this lack of information represents a critical gap in our understanding of trypanosome cell biology.

TABLE I
Conditions used for immunofluorescence with ␣-TLTF antibodies Antibodies were raised in rabbits, affinity-purified, and used for immunofluorescence as described under "Experimental Procedures." The TLTF amino acids (aa) included in the antigens used for preparation of each antiserum are indicated in parentheses. Antibodies were used undiluted or at dilutions of 1:10 through 1:1000 (TT1 and TT4) or 1:10 through 1:3000 (Pep4). Primary antibody incubation times ranged from 30 min at room temperature to 24 h at 4°C. Trypanosome cytoskeletons and isolated flagella were prepared as described under "Experimental Procedures." Fixations containing methanol were done for 20 min at Ϫ20°C. All other fixations were done for both 30 and 60 min at room temperature. Blocking incubations ranged from 30 min at room temperature to 24 h at 4°C. Permeabilizing and quenching agents were employed for samples fixed with aldehyde reagents. SDS treatments were done for 5 min at room temperature in PBS after fixation and samples were blocked after SDS treatment.
In addition to the flagellar axoneme, the other predominant structure in the flagellum is the paraflagellar rod (PFR), a lattice-like filament composed of two major protein subunits, PFR-A and PFR-C (2). The PFR is contained within the flagellar membrane and is attached to axoneme by filamentous cross-links (Refs. 2 and 12; Fig. 7). The flagellum is attached to the cytoplasmic cytoskeleton in a flagellum attachment zone that runs from the flagellar pocket to the anterior end of the cell (Refs. 1, 2, and 12; Fig. 7). The cytoplasmic side of the flagellum attachment zone is defined by an electron-dense "FAZ filament" that originates at the anterior side of the flagellar pocket and extends to the anterior end of the cell (Fig. 7).
Immediately adjacent to the FAZ filament is a quartet of specialized microtubules, which, unlike other microtubules of the subpellicular corset, do not extend to the posterior side of the flagellar pocket (3). These four microtubules are further distinguished by their association with a membranous tubule (12). The PFR, FAZ filament, and quartet of reticulum-associated microtubules remain intact and attached to the flagellum after detergent and Ca 2ϩ extraction (12), as does a "tripartite attachment complex" that links the flagellar basal body apparatus with the kinetoplast (2,5). Our data indicate that TLTF is a component of one of these Ca 2ϩ -resistant, flagellum-associated cytoskeletal complexes. Together, these complexes function in several fundamental cellular activities, including cell motility, cytokinesis, and organelle inheritance (2). Since immunofluorescence studies have not allowed us to distinguish between these possible functions for TLTF, we are currently working to determine whether disruption of TLTF gene expression compromises any of these processes.
Western blot analysis of total trypanosome protein extracts with ␣-TLTF-TT1 antibodies detected a 41-kDa protein that is present in procyclic trypanosomes and absent in bloodstream parasites (Fig. 1A). Although this 41-kDa protein is distinct from TLTF, it appears to share an epitope with TLTF that is recognized by some, although not all, ␣-TLTF antibodies in FIG. 5. Multiple sequence alignment of TLTF and TLTF-related proteins from other organisms. A, alignment of amino acids 33-247 of TLTF from T. brucei (Tb) and the corresponding sequences of related proteins from humans (Hs), zebrafish (Dr), S. mansoni (Sm), D. melanogaster (Dm), and T. cruzi (Tc). Amino acids that are identical in three or more proteins are shaded dark gray, and conservative substitutions are shaded light gray. Asterisks mark positions that are identical in all proteins for which the sequence is known. Numbering is based on the full-length TLTF amino acid sequence (15). B, alignment of amino acids 269 -429 of TLTF from T. brucei (Tb) and the corresponding sequences of related proteins from humans (Hs) and C. reinhardtii (Cr). Amino acids that are identical in two or more proteins are shaded dark gray, and conservative substitutions are shaded light gray. Asterisks mark positions that are identical in all proteins for which the sequence is known. Numbering is based on the full-length TLTF amino acid sequence (15). Complete sequences are known only for the T. brucei (15) and human (40) proteins. The sequences of these full-length proteins are 34% identical and 60% similar (not shown). Also not shown is a mouse protein that is 87% identical to the human protein (15). DNA sequences encoding partial TLTF-like proteins were identified by BLAST searches of the GenBank TM nonredundant and dbEST data bases. Accession numbers for these sequences are as follows: zebrafish (AI584969), T. cruzi (AQ444061), D. melanogaster (AAF45877), schistosomes (AI067540), and C. reinhardtii (BE056668). Amino acid sequence alignments were done using the multiple sequence alignment algorithm of the Wisconsin GCG sequence analysis software package.
Western blots (Figs. [1][2][3][4][5]. Since this 41-kDa protein is not recognized by antibodies directed against either the N-terminal 145 amino acids (TT4) or the C-terminal 14 amino acids (Pep4) of TLTF, this shared epitope most likely occurs within amino acids 141 and 439 of TLTF. The developmental stage-specific expression of this 41-kDa protein suggests that it functions in a process that is specific to procyclic trypanosomes in the insect host environment.
Comparison with Other Cytoskeleton-associated Proteins from T. brucei-Over the last several years, other nontubulin cytoskeletal proteins have been identified in T. brucei (4). Many, although not all (e.g. see Ref. 45), of these proteins are associated with the subpellicular cytoskeleton, rather than with the flagellar fraction of the cytoskeleton. A general feature of these proteins is that they are usually quite large (molecular mass Ն120 kDa) and possess highly repetitive amino acid sequences. Balaban et al. (13) employed biochemical fractionation to isolate an unidentified 52-kDa protein that co-purifies with intact trypanosome cytoskeletons and induces microtubule bundling in vitro. While similar in mass to TLTF, this FIG. 6. The human TLTF-like protein (GAS11) contains a domain that co-localizes with microtubules in mammalian cells. COS-7 cells were transfected with a plasmid encoding a GFP fusion protein containing amino acids 115-258 of the human GAS11 protein (Fig. 5) and subjected to immunofluorescence using a monoclonal antibody against ␤-tubulin as described under "Experimental Procedures." Cells were untreated (A-H) or treated (I) with 10 M nocodazole for 4 h prior to fixation and immunofluorescence. Cells in A-H were extracted with 0.5% Triton X-100 prior to fixation. Cells were examined by laser-scanning confocal fluorescence microscopy. Fluorescent images of GFP (A and D) or tubulin (B and E) were captured on separate channels and then merged (C, F, and G-I). Note that these cells were transiently transfected; hence, not all tubulin-stained cells express GFP. H shows an enlargement of the boxed region in G. The GAS11-GFP fusion protein exhibits a punctate distribution along a subset of microtubules and tends to accumulate at microtubule ends or gaps (e.g. arrowheads in H).
52-kDa protein is released from the cytoskeleton by depolymerization of the subpellicular cytoskeleton and is therefore distinct from TLTF.
Additional T. brucei cytoskeletal proteins have been detected using antibodies raised against whole cytoskeletons (14). While the functions and identities of most of these proteins remain to be determined, most are quite large (molecular mass Ն120 kDa) and are usually associated with the subpellicular cytoskeleton or, in some cases, the flagellum/flagellum attachment zone (14). One exception is the BBA4 antigen, which exhibits a punctate localization at the flagellar basal body apparatus (14). This localization pattern is similar to what we see for a TLTF-GFP fusion protein (15,17) and is also observed for TBBC, another coiled-coil protein of T. brucei (45). Interestingly, TBBC was originally isolated using antibodies directed against an immunosuppressive factor from T. brucei (45). This is reminiscent of our initial studies with TLTF, which was first isolated on the basis of its immunomodulatory activity (15,46). Although recombinant TLTF is capable of stimulating T cells (15), such an activity seems to be at odds with its intracellular association with the trypanosome cytoskeleton. While this apparent discrepancy remains to be resolved, the coincidental finding of another coiled-coil cytoskeletal protein (i.e. TBBC) that is antigenically related to an immunomodulatory protein may suggest a general feature of these proteins. Perhaps the ability of TLTF to activate T cells is related to the presence of coiled-coil domains and the general capacity of cytoskeletal proteins to engage in higher order protein-protein complexes. Alternatively, perhaps the apparent multiplicity of functions ascribed to TLTF stems from the presence of other trypanosome proteins with epitopes that are related to TLTF (e.g. see Fig. 1A).
TLTF is distinguished from most cytoskeletal proteins thus far identified in T. brucei, since it is small by comparison and lacks the multiple direct repeats often seen in these proteins. However, TLTF is predicted to contain extensive ␣-helical secondary structure and to form multiple coiled-coils (17) (6). B, idealized transverse section taken through the midpoint of the cell, looking from posterior to anterior. The flagellum (top) contains a typical set of "9 ϩ 2" axoneme microtubules (7) together with a filamentous structure called the PFR (6). The PFR is connected to the axoneme and to an electron-dense filament (5) in the cytoplasm by regularly spaced cross-bridges. Immediately adjacent to this cytoplasmic filament is a specialized quartet of microtubules (3) that are subtended by a membranous tubule (4). These reticulum-associated microtubules, together with the cytoplasmic filament (5), make up a FAZ that runs from the flagellar pocket to the anterior end of the cell (2). C, cut-out view of the flagellar pocket area, emphasizing the cytoskeletal structures discussed in the Introduction and under "Results" and "Discussion." The flagellar pocket (9) is formed by an invagination of the plasma membrane where the flagellum emerges from the basal body complex (8). The plasma membrane and flagellar membrane are held in close juxtaposition by electron-dense adhesion zones (10). The cytoplasmic components of the flagellum attachment zone (3, 4, and 5) extend from the flagellar pocket to the anterior end of the cell. Most subpellicular microtubules (4) extend the entire length of the cell. However, the specialized quartet of FAZ microtubules (3) does not extend to the posterior side of the flagellar pocket. Although the precise location of the ends of these four microtubules is not known, they might originate from the basal body complex (dashed lines), which would give them an inverse orientation relative to the other subpellicular microtubules (2). This quartet of microtubules, together with the axoneme, paraflagellar rod, FAZ filament, and basal body complex, are resistant to detergent and Ca 2ϩ extraction (30,34) and comprise the flagellar fraction seen in lane P2 of Fig. 4. The arrow points to the position of a GFP fusion protein containing a minimal TLTF targeting domain of amino acids 114 -257, as determined by immunoelectron microscopy (17). These illustrations are based on our observations, as well as the published electron microscopy studies of K. Vickerman (39) and K. Gull (12). 1, flagellum; 2, subpellicular microtubules; 3, special quartet of reticulum-associated microtubules; 4, microtubule-associated reticulum; 5, flagellum attachment zone (FAZ) filament; 6, PFR; 7, axoneme; 8, basal body complex; 9, flagellar pocket; 10, adhesion zones. somes (33) and in other organisms (38). Supporting this observation is the fact that PSI-BLAST (47) searches of GenBank TM identify several myosin sequences as having TLTF alignment E (expect) values that exceed the threshold (not shown).

A Candidate Human Tumor Suppressor Is Related to TLTF and Contains a Domain That Co-localizes with Microtubules in
Mammalian Cells-The mammalian growth arrest-specific protein, GAS11, has extensive sequence similarity to TLTF (Fig. 5) (15, 17). The human GAS11 gene is expressed in a variety of human tissues (40) 2 and is located on chromosome 16, in a region (16q24.3) whose deletion is associated with breast and prostate cancer (40). Although a role in breast cancer has now been ruled out, GAS11 remains a candidate tumor suppressor gene for prostate cancer (40). The important role played by the cytoskeleton in preventing uncontrolled growth of tumors is evidenced by the growing number of anticancer drugs that act by stabilizing microtubules (48). Our finding that TLTF is a component of the trypanosome cytoskeleton makes it tempting to speculate that GAS11 plays a role in the organization and/or function of the cytoskeleton in mammalian cells. Consistent with this possibility, human GAS11 contains a domain that directs a GFP fusion protein to the plus-end of microtubules in trypanosomes (17). This same fusion protein co-localizes precisely with microtubules in mammalian cells (Fig. 6). Whether this co-localization is through direct or indirect interaction with microtubules remains to be determined. Interestingly, the GAS11-(115-258)-GFP fusion protein does not decorate all microtubules and tends to be localized to ends or gaps in microtubules (arrowheads in Fig.  6H). This patchy distribution along a subset of microtubules is distinct from what is observed for microtubule-associated proteins such as Tau and MAP2c (49) but is very reminiscent of the localization pattern exhibited by the N-terminal microtubulebinding domain of CLIP-170, which directs GFP to the plus-end of microtubules in mammalian cells (24,42). CLIP-170 is a cytoplasmic linker protein implicated in regulating dynamic assembly of microtubules (43). Growth arrest-specific genes have been implicated a variety of functions, including downregulation of cell growth (50) and organization of the actin cytoskeleton (51). Since the cytoskeleton plays a central role in controlling cell growth, a cytoskeletal milieu for GAS11 is consistent with its up-regulation in growth-arrested cells. 3 The TLTF-related sequences in genome data bases of mice (15), humans, zebrafish, T. cruzi, S. mansoni, D. melanogaster, and C. reinhardtii (Fig. 5) contain long stretches of nearly identical amino acid sequences but possess enough differences to suggest that they are distinct proteins and may have related but not identical functions. Likewise, the insect form-specific 41-kDa protein of T. brucei that contains a shared epitope with TLTF, within residues 141-439, might fall into this group of proteins. Hence, our results indicate that TLTF is the founding member of a new family of cytoskeletal proteins present in organisms as diverse as protozoa and humans. Repeated searches of the S. cerevisiae genome data base have not revealed any TLTF-like genes, perhaps reflecting a fundamental difference in the cytoskeleton organization of budding yeast compared with trypanosome and mammalian cells.