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1 Environmentally-regulated glycosome protein composition in the African 2
trypanosome 3 Sarah Bauer, James C. Morris, Meredith T. Morris# 4 Department of Genetics and Biochemistry, Clemson University, Clemson, SC 29634 5 Running Title: Glycosome heterogeneity in T. brucei 6 7 8 9
# Corresponding author: Dept. of Genetics and Biochemistry, Clemson University, 217 BRC, 10 51 New Cherry Street, Clemson SC 29634. Tel. (864) 656-0367, FAX: (864) 656-0393; 11 Email: [email protected]
Copyright © 2013, American Society for Microbiology. All Rights Reserved.Eukaryotic Cell doi:10.1128/EC.00086-13 EC Accepts, published online ahead of print on 24 May 2013
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ABSTRACT: 13 14 Trypanosomes compartmentalize many metabolic enzymes in glycosomes, 15 peroxisome-related microbodies that are essential to parasite survival. While it is 16 understood that these dynamic organelles undergo profound changes in protein 17 composition throughout life cycle differentiation, the adaptations that occur in 18 response to changes in environmental conditions are less appreciated. We have 19 adopted a fluorescent-organelle reporter system in procyclic T. brucei by expressing 20 a fluorescent protein (FP) fused to a glycosomal targeting sequence (PTS2). In these 21 cell lines, PTS2-FP is localized within import competent glycosomes and organelle 22 composition can be analyzed by microscopy and flow cytometry. Using this 23 reporter system, we have characterized parasite populations that differ in their 24 glycosome composition. In glucose-rich media two parasite populations are 25 observed; one population harbors glycosomes bearing the full repertoire of 26 glycosome proteins while the other parasite population contains glycosomes that 27 lack the usual glycosome resident proteins but do contain the glycosome membrane 28 protein TbPEX11. Interestingly, these cells lack TbPEX13, a protein essential for the 29 import of proteins into the glycosome. This bimodal distribution is lost in low-30 glucose media. Furthermore, we have demonstrated that changes in 31 environmental conditions trigger changes in glycosome protein composition. These 32 findings demonstrate a level of procyclic glycosome diversity heretofore 33 unappreciated and offers a system by which glycosome dynamics can be studied in 34
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live cells. This work adds to our growing understanding of how the regulation of 35 glycosome composition relates to environmental sensing. 36 37 38
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INTRODUCTION 39 Trypanosoma brucei, the causative agent of human African trypanosomiasis, has a 40 complex life cycle, with developmental stages in the bloodstream of the mammalian 41 host and the tsetse fly vector. Each host provides a distinct environment in which 42 the parasites must survive. Bloodstream form (BSF) parasites are bathed in glucose 43 and generate ATP exclusively by glycolysis. While in the tsetse fly, the procyclic 44 form (PF) parasites experience a drop in glucose levels with a concomitant increase 45 in the availability of amino acids (namely proline). Under these conditions, the 46 parasite adapts its metabolism, generating ATP from both glycolysis and amino acid 47 metabolism (1). 48 49 In trypanosomes, many of the enzymes involved in glycolysis are contained within 50 membrane-bounded organelles called glycosomes (reviewed in (2-4)). Similarities 51 between the metabolic activity and the matrix protein import machinery of 52 glycosomes and peroxisomes indicate an evolutionary relationship between the two 53 organelles. In contrast to peroxisomes, however, glycosomes are essential, making 54 mechanisms of glycosome biogenesis and maintenance attractive drug targets. 55 56 Glycosome dynamics are governed by a number of processes including organelle 57 biogenesis, protein import, and changes in protein composition. In trypanosomes, a 58 number of proteins involved in import have been characterized and recent studies 59 have begun to identify processes involved in glycosome turnover and remodeling 60 (5-7). However, very little is known about organelle biogenesis. 61
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62 Proper regulation of glycosome number and composition is essential to T. brucei. 63 Silencing of trypanosome PEX proteins involved in glycosome matrix protein import 64 (TbPEX5, 7, 10, 6, 12, 14) causes the mislocalization of glycosome proteins to the 65 cytosol and compromised growth (8-12). Reduction in the expression of PEX genes 66 involved in glycosome biogenesis (TbPEX11and TbPEX19) through RNA 67 interference (13, 14), results in parasites harboring fewer, larger glycosomes, while 68 overexpression of TbPEX11 results in cells with many smaller glycosomes (14). As 69 observed after silencing of other PEX genes, reduction in levels of TbPEX11 and 70 TbPEX19 also resulted in growth arrest. 71 72 T. brucei glycosomes are extensively remodeled during differentiation between BSF 73 and PF parasites. In a recent study, 159 proteins from the glycosomes of BSF and PF 74 were identified by proteomics (15). Of these proteins, approximately 35% were 75 found in both stages of the life cycle. These constitutively expressed proteins 76 included enzymes involved in glycolysis, purine salvage, pyrimidine biosynthesis, 77 phospholipid degradation and glycerol-ether lipid biosynthesis. Forty-two percent 78 of the proteins were PF-specific and included proteins involved in the pentose 79 phosphate pathway, the Calvin-Benson cycle and oxygen radical and peroxide 80 detoxifying enzymes. 81 82 While it is understood that the metabolic repertoire of the glycosome is variable, the 83 details of glycosome biogenesis and changes in protein composition are unknown. 84
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Recent work suggests that glycosomal proteins can be turned over during the 85 differentiation process in a pathway that is mechanistically analogous to autophagy 86 (16). Glycosomes colocalize with lysosomes during differentiation when there is a 87 change in glycosome protein expression. Autophagy of glycosomes may also play a 88 role in the parasites’ response to the environment, as this pathway appears to be 89 induced under starvation conditions when parasites are moved from nutrient-rich 90 culture to PBS (16, 17). PBS-induced autophagy is not restricted to T. brucei, but has 91 also been reported in the related kinetoplastid, Leishmania major (18). In contrast 92 to our knowledge of the changes that occur during the differentiation process, less is 93 known about how the protein composition of PF glycosomes changes in response to 94 different environmental conditions. 95 96 In yeast and mammalian cells, peroxisomes can proliferate through the growth and 97 division of existing organelles as well as through de novo synthesis from the 98 endoplasmic reticulum. Aside from the observations that the silencing and 99 overexpression of TbPEX11 leads to defects in glycosome number and morphology, 100 nothing is known about how glycosomes proliferate through the growth and 101 division of existing organelles and, to our knowledge, a de novo pathway of 102 glycosome biogenesis in T. brucei has not been examined. Searches of the 103 trypanosome genome have failed to identify any homologs of genes involved in de 104 novo biogenesis, however, this is not surprising as these proteins are not well 105 conserved and trypanosome sequences are highly divergent from higher eukaryotes. 106 107
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To study glycosome dynamics, we have adopted a fluorescent-organelle reporter 108 system used to study peroxisome biogenesis in yeasts and mammals for use in 109 procyclic form T. brucei. In this system, mature glycosomes import glycosomally-110 targeted fluorescent protein while immature organelles do not. Using flow 111 cytometry and electron microscopy, we have identified and characterized two 112 parasite populations that differ in their glycosome composition. One population 113 contains “mature” glycosomes harboring the expected repertoire of glycosomal 114 proteins including TbPEX13, a protein essential for import of proteins into 115 glycosomes, while the other population contains glycosomes, as demonstrated by 116 EM, but expressed little or no glycosome proteins including TbPEX13. The relative 117 proportions of each parasite population varied in different media conditions. In the 118 presence of glucose, two distinct populations were present; one “dim” population 119 harboring immature glycosomes and one “bright” population harboring mature 120 glycosomes, with very few cells of intermediate fluorescence observed. 121 Interestingly, in low-glucose media this bimodal population structure was lost and a 122 range of cells with different fluorescence intensity was observed. When parasites 123 were moved from low-glucose media into media containing 5 mM glucose, cells of 124 intermediate fluorescence were lost within 24 h. In addition to changes in steady 125 state glycosome composition under different media conditions, changes in 126 glycosome expression as monitored by PTS2-FP expression was observed in live 127 cells during changes in environmental conditions. This change was initiated within 128 3 h of cell passage from log-phase culture to fresh media and was complete within 129 24 h. 130
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131 The results presented here demonstrate a level of glycosome diversity previously 132 unrecognized. Furthermore, this is the first demonstration that PF parasites harbor 133 immature glycosomes and provides the first suggestion that T. brucei glycosomes 134 may proliferate via a de novo pathway. The glycosome reporter system utilized in 135 these studies provides a rapid, high-throughput, real-time protocol to monitor 136 processes such as autophagy, which likely regulate glycosome remodeling, in live 137 cells.138
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MATERIALS AND METHODS 139 Reagents: All reagents were purchased from Fisher Scientific unless specified. 140 141 Growth and transfection of parasites: T. brucei 29-13 procyclic form parasites, 142 which express T7 RNA polymerase as well as the tetracycline repressor, were 143 maintained in SDM-79 as described (19). PF-PTS2-FP previously named pXS2-144 Aldo-PTS-eYFP (20) and PF-FP were maintained in SDM-79 containing G418 145 (15µg/ml), hygromycin (50µg/ml) and blasticidin (15µg/ml). Clonal cell lines were 146 obtained by limiting dilution (~0.33 parasites/well) into 96 well plates. 147 148 Generation of fluorescent reporter strains: To generate PF-FP parasites, the open 149 reading frame of green fluorescent protein (GFP) was cloned using the HindIII site 150 into the pXS2 (21) (kind gift provided by J. Bangs, University of Wisconsin) 151 expression vector in which the neomycin resistance gene was replaced with the 152 blasticidin resistance gene (pXS2bla). This plasmid integrates into the tubulin locus 153 and constitutive expression is driven by the PARP promoter. Orientation was 154 confirmed by sequencing. PF parasites were stably transfected as described (22) 155 with 10 µg of the MluI linearized pXS2-FP construct and selected by supplementing 156 the growth media with 15 µg/mL blasticidin. 157 158
Live cell microscopy: For analysis of fluorescence in live cells, parasites were 159 collected by centrifugation (800 x g, 10 min.) and washed once with PBS. Cell 160 pellets were then resuspended in vectashield mounting media (Vector laboratories, 161
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Burlingame, CA) and visualized on a Zeiss Axiovert 200M using Axiovision software 162 version 4.6.3 for image analysis. 163 164 Transmission electron microscopy. For ultrastructural analysis, PF parasites 165 were fixed in 2% paraformaldehyde/2.5% glutaraldehyde in 100 mM phosphate 166 buffer for 1 hr at room temperature. Samples were washed in phosphate buffer and 167 postfixed in 1% osmium tetroxide (Polysciences Inc., Warrington, PA) for 1 hr. 168 Samples were then rinsed extensively in dH20 prior to en bloc staining with 1% 169 aqueous uranyl acetate (Ted Pella Inc., Redding, CA) for 1 hr. Following several 170 rinses in dH20, samples were dehydrated in a graded series of ethanol and 171 embedded in Eponate 12 resin (Ted Pella Inc.). Ultrathin sections of 90 nm were 172 obtained with a Leica Ultracut UCT ultramicrotome (Leica Microsystems Inc., 173 Bannockburn, IL), stained with uranyl acetate and lead citrate, and viewed on a JEOL 174 1200 EX transmission electron microscope (JEOL USA Inc., Peabody, MA) at the 175 Molecular Microbiology Imaging Facility, Washington University School of Medicine 176 in St. Louis. 177 178 Glycosome measurements: ImageJ software (http://rsb.info.nih.gov/ij/) was 179 used to analyze electron microscopy images of both bright and dim parasite 180 populations. The area of cell visible and individual glycosomes were measured for 181 each image. To calculate glycosome density, the total number of glycosomes was 182 divided by the area of cell visible. This value was then converted to 183 glycosomes/100μm2. To calculate the percent of cell area occupied by glycosomes, 184
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the area of all glycosomes was summed and divided by the area of cell visible, for 185 each image analyzed. These values were averaged for each population. From the 186 dim population , 13 images, containing 24 cells and 87 glycosomes were analyzed. 187 From the bright population, 13 images, 26 cells and 119 glycosomes were analyzed. 188 189 Cytometric analysis, cell sorting and western blot analysis: Enhanced YFP 190 fluorescence in live cells was monitored using either a BD FACSCan flow cytometer, 191 Accuri C6, or BD Influx cell sorter. Fluorescence emission at 530/540 nm (FITC 192 channel) was collected after excitation with a 488 nm laser and data were analyzed 193 using FLOWJO software (TreeStar Inc., Ashland, OR). Cells were sorted directly into 194 SDM-79 using the BD Influx with a 100 micron tip at a sheath pressure of 12 psi and 195 a drop frequency of 28.7 kHz and samples were processed for EM and western 196 analysis immediately. Cell viability after sorting was confirmed by microscopy and 197 estimated (by counting of live cells) to be > 90%. For western blotting, cell lysates 198 (5x 106-107 cells) were resolved by 12% SDS-PAGE and transferred to Protran 199 nitrocellulose. Blots were processed as described in (23) and probed with the 200 following antibodies: TbHK (1:10,000), PFK (1:10,000), FPBase (1:10,000), TIM 201 (1:10,000), G6PDH (1:10,000), GK (1:10,000), PEX 13 (1:10,000), which were 202 provided by Paul Michels (de Duve Institute and Université catholique de Louvain, 203 Brussels (9)). Rabbit anti-glycosome antibodies (1:1000) 2841D were provided by 204 Marilyn Parsons ((24) SBRI). TbPEX11 (1:4,000) antibodies were provided by 205 Christine Clayton (14)(Universität Heidelberg, Heidelberg, Germany). Mouse anti-206 GFP antibodies (1:1,000, Molecular Probes, Eugene, OR), were used to detect eYFP. 207
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208 Analysis of environmentally-dependent changes in glycosome composition: 209 Cells cultured for extended periods of time are diminished in their ability to respond 210 to environmental changes. Therefore, immediately after transformants emerge 211 from drug selection, stable cell lines are stored in freezing media (24mM KCl, 0.03 212 mM CaCl2, 2 mM K2HPO4, 5 mM HEPES, 0.4 mM EDTA, 1 mM MgCl2 in 50% glycerol) 213 in LN2. Before use, cells were thawed and seeded at a density of 1x105/ml. When 214 cells reached log-phase, ~6 x 106/ml, they were passed into fresh media to a final 215 concentration of 1 x 105/ml or 5 x 105/ml and analyzed by flow cytometry over 24 h. 216 Prior to each remodeling assay, cells were seeded to a density 1x105/ml and grown 217 to log-phase. After transformation, cells were passaged no more than twice after 218 which they were decontaminated and discarded and new cells are transformed. 219 220 on M
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RESULTS 221 222 Cytometric analysis reveals two cell populations that differ in their relative 223 fluorescence. We have previously generated a fluorescent glycosomal reporter 224 system for use in live cells by fusing the peroxisome targeting sequence 2 (PTS2) of 225 aldolase, which targets proteins to the glycosome (25), to the open reading frame of 226 eYFP (FP). In this cell line, PF-PTS2-FP (previously named 29-13-pXS2-Aldo-PTS-227 eYFP), constitutively expressed PTS2-FP is targeted to glycosomes as indicated by 228 fluorescence microscopy (Fig. 1A and (20)). 229 230 Flow cytometry of PF-PTS2-FP cultures revealed two distinct populations (one 231 “bright” and one “dim”) that differed by almost two orders of magnitude in relative 232 fluorescence (Fig. 1B). This was in contrast to PF-FP cells expressing GFP lacking a 233 PTS2 signal sequence (PF-FP, Fig. 1B), which yielded a single peak. Both bright and 234 dim cell populations in the PF-PTS2-FP cultures excluded propidium iodide equally, 235 suggesting that viability was not compromised in the cells with reduced 236 fluorescence (data not shown). Four independent clonal cell lines obtained by two 237 separate transformations were analyzed and all contained both bright and dim 238 populations, further suggesting that the differences in expression were not due to 239 unexpected integration events or artifacts introduced via selection. 240 241 Cells of different fluorescent intensities differ in glycosome protein 242 composition but have similar glycosome morphologies. 243
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To explore the absence of PTS2-FP in the dim cells and directly assess the 244 expression of other glycosomal proteins, both bright and dim populations were 245 analyzed by western blotting. Cells were sorted according to their relative 246 fluorescence intensities and sorting efficiencies were confirmed by cytometric 247 analysis of the sorted populations (Fig. 2A). Lysates from equal cell equivalents (5 x 248 105) of each population were then analyzed by western blotting using antibodies 249 that recognize eYFP, and antisera (2841D, (24) that recognize the two glycosomal 250 matrix proteins, aldolase and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). 251 Notably, the cells from the dim population lacked detectable levels of PTS2-FP, 252 aldolase and GAPDH but expressed equivalent levels of the surface protein, 253 procyclin (Fig. 2B). Further western analysis was used to assess the expression of 254 other glycosomal proteins in bright and dim cells (Fig. 2C). Dim cells expressed low 255 or undetectable levels of TbHK, FPBase, TIM, G6PDH, and glycerol kinase. 256 Interestingly, TbPEX11, an integral glycosome protein was detected in dim cells, 257 while a protein essential to glycosome protein import, TbPEX13, is not detectable. 258 These findings suggest that dim cells harbor glycosomes but that they are not 259 competent to import matrix proteins. 260 We next used transmission electron microscopy to confirm the presence of 261 glycosomes in dim cells as well as analyze morphology of these organelles in the 262 different cell populations (Dim, Fig. 3A; Bright, Fig. 3B). In both bright and dim cells, 263 electron dense glycosomes were observed. There was no dramatic difference in size, 264 number or location of glycosomes in each of the populations. Bright cells had an 265 average of 112 glycosomes/100μm2 cell area (Fig. 3E), with an average area of 0.02 266
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μm2 (Fig. 3C) and made up 2% of the cell area (Fig. 3D). Average glycosome number 267 in dim cells was 119 cells/100μm2 (Fig. 3E), with an average area/glycosome of 268 0.027μm2 (Fig. 3C) and comprised 2.43% of the area of the cell (Fig. 3D). 269 270 Cells of intermediate fluorescence are observed in low glucose media. Dim 271 cells have glycosomes, as demonstrated by EM. However, the lack of TbPEX13 272 suggested that these glycosomes were not able to import glycosomal proteins. It is 273 known that the mislocalization of glycosomal proteins is lethal when cells are grown 274 in standard SMD-79 with glucose. We therefore hypothesized that cells harboring a 275 large number of immature glycosomes must repress glycosome protein expression 276 under standard culturing conditions. In the absence of glucose, the mislocalization 277 of glycosome proteins is less detrimental to the parasites and we reasoned that 278 under these conditions, cells of intermediate fluorescence would be detected. To 279 test this, we grew cells in low-glucose media (SDM-80), which was used previously 280 to study how PF metabolism changes in response to extracellular levels of glucose 281 and proline (26), and monitored fluorescence by flow cytometry (Fig. 4). Indeed, 282 when cultures were grown in low-glucose conditions, cells of intermediate 283 fluorescence were observed, resulting in a single broad peak consisting of cells 284 exhibiting a continuous range of fluorescence intensities. Furthermore, addition of 285 glucose (5mM) to SDM-80 restored the bimodal population distribution indicating 286 that glucose is the media component responsible for these differences in glycosome 287 composition (Fig. 4). 288 289
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We next measured the rate at which this intermediate population changed in 290 response to different media conditions. Cells grown in SDM-80 were passed into 291 SDM-79 and analyzed by flow cytometry. A decrease in the number of intermediate 292 cells was observed at one hour and by 24 h two distinct populations were observed. 293 In contrast, when cells were moved from SDM-79 into SDM-80, it took longer for the 294 intermediate population to appear (Fig. 5). By day 5 a continuous range of cells with 295 varying levels of fluorescence intensities was observed. Because cells harboring 296 immature glycosomes are so well resolved in SDM-79, we have chosen to use these 297 conditions to study changes in glycosome composition that occur in response to 298 changing environmental conditions. 299 300 Changes in media conditions trigger changes in glycosome protein expression. 301 In yeast and mammalian cells, peroxisome turnover is triggered by changes in 302 media conditions (27). To test the effect that environmental changes have on 303 glycosome composition, we passed cells from a log-phase culture into fresh SDM-79 304 at a concentration of 105/ml bright and dim measured populations by flow 305 cytometry. When cells were passed from log-phase culture to fresh media at a 306 concentration of 1 x 105/ml, the number of cells falling within the left gate (includes 307 dim cells exhibiting background fluorescence as well as cells of intermediate 308 fluorescence) increased 3-fold with 30.1% dim cells present at 3 h. post-passage 309 (Fig.6B) as opposed to 10.8% of cells in the left gate in log-phase culture (Fig. 6A) . 310 By 24 h., the original population distribution was restored (Fig. 6C). When cells 311 were subjected to a less dramatic change in cell number and media conditions by 312
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passing them to a final concentration of 5 x 105/ml there was no change in 313 population distribution (Fig. 6 D and E). 314 315
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DISCUSSION 316 317 We have generated a fluorescent glycosome reporter system in T. brucei that allows 318 us to quickly visualize glycosomes in large numbers of live cells under a variety of 319 different conditions. Using this system, we have identified two populations of PF 320 parasites in culture that harbor different glycosomes. This work reveals that PF 321 glycosome composition varies in an environmentally-dependent manner (Fig. 7). 322 323 Dim cells express very little or undetectable levels of a number of glycosome matrix 324 proteins. TEM showed that dim cells do harbor glycosomes; a finding that is 325 consistent with the expression of the integral glycosome membrane protein, 326 TbPEX11, in these cells. These results, along with the absence of TbPEX13, a protein 327 involved in glycosome protein import, suggested that dim parasites harbor 328 immature glycosomes that are unable to import glycosome proteins. We propose 329 that because these organelles are import incompetent and because mislocalization 330 of glycosomal proteins is lethal under most conditions (12, 28), the expression of 331 glycosomal proteins must be repressed until enough mature organelles are present 332 to correctly localize glycosomal matrix protein. We hypothesize that this tight 333 regulation of glycosome protein is responsible for the bimodal population 334 distribution observed in standard SDM-79 media. Consistent with this hypothesis is 335 the observation that when cells are grown in low-glucose conditions in which the 336 mislocalization of glycosome proteins is tolerated, cells with intermediate 337 fluorescence intensities are observed. 338
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339 Several lines of evidence argue against the supposition that these observations are a 340 consequence of misregulated expression of recombinant fluorescent protein in a 341 transgenic system. First, it should be noted that the expression of cytosolic GFP 342 from the same expression vector (pXS2 derivative) yielded a single homogenous 343 population, indicating that the bimodal population is likely not a consequence of 344 unidentified regulation signals in the expression constructs or integration into 345 unanticipated sites in the genome. Furthermore, the dim cells lack not only the 346 PTS2-eYFP reporter, but also aldolase and GAPDH as well as a number of other 347 glycosome proteins. It is unlikely that transformation and expression artifacts 348 would affect expression of proteins other than PTS2-FP. 349 350 De novo biogenesis of peroxisomes has been demonstrated in yeast and mammalian 351 cells ((29, 30). In this process, the sequential maturation of peroxisomes is 352 observed whereby pre-peroxisomal vesicles bud from the ER. In S. cerevisiae, the 353 peroxin, PEX3, was fused to yellow fluorescent protein and found to localize to 354 immature pre-peroxisomal vesicles which bud from the ER. The protein first 355 localized to the ER with another peroxin, PEX19. PEX3-PEX19 containing foci were 356 then found to bud off from the ER and newly formed peroxisomes capable of 357 importing matrix proteins, developed into mature glycosomes or fused with existing 358 organelles (31). Another example of the temporal regulation of peroxisome protein 359 import is seen in the yeast Hansenula polymorpha. In this model system, 360 peroxisomes first grow by the incorporation of lipids and proteins. After reaching a 361
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threshold size, the organelles give rise to new ones through fission. The progenitor 362 organelle is metabolically active, but no longer capable of importing new protein (32, 363 33). To our knowledge the presence of a de novo biogenesis process or sequential 364 glycosome maturation in T. brucei has never been examined and to date, database 365 analysis has failed to reveal a PEX3 homolog. 366 367 We found that changes in glycosome composition occur rapidly in response to 368 changes in environmental conditions. Passage of cells from a log-phase culture to 369 fresh media triggered a transient increase in the percentage of “dim” cells that fell 370 within the left gate. This change was initiated within 3 h and the original population 371 distribution was restored within 24 h. We find that the manner in which the cells 372 are cultured dramatically affect their ability to respond to changes in the 373 environment. In some cases, this increase in the “dim” population approached 374 100%, while cells cultured for long periods of time become insensitive to changes in 375 the environment (data not shown). To control for this, cells used in remodeling 376 assays are thawed from stabilates and seeded at 1 x105/ml and grown to a density 377 of 6x106/ml before being passed into fresh media. Cells used in the assays are not 378 passed more than twice. We have found that these conditions provide the most 379 reproducible, although not always the most dramatic, results. Changes in cellular 380 metabolism as well as changes in media composition that occur during growth in 381 culture likely contribute to the variability of the cells response to changes in 382 environment. We are currently in the process of identifying and characterizing the 383
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effect of different variables on changes in glycosome composition as we believe this 384 is important to understanding the biological relevance of such responses. 385 386 The time-frame of these changes in glycosome composition is consistent with the 387 process of autophagy. To date, we have not been able to demonstrate any effect of 388 autophagy inhibitors such as 3-methyladenine and wortmannin on remodeling. 389 However, it is difficult to determine if these compounds work to inhibit autophagy 390 under our experimental conditions. We are in the process of assessing the efficacy 391 of such inhibitors in blocking autophagy under our conditions. Furthermore, we are 392 using TEM to assess the extent to which autophagic structures are formed during 393 this remodeling process. 394 395 Dilution of cells from a log-phase culture into fresh media changes the carbon 396 source availability and triggers a change in glycosome composition. Peroxisome 397 remodeling in response to environmental conditions is documented in a number of 398 organisms. For example, in S. cerevisiae, changes in peroxisome composition are 399 observed when cells are moved from an oleic acid based media to one in which 400 glucose is included as a carbon source (34). Under these conditions, the peroxisome 401 matrix protein, Fox3p, is degraded. In methanol media, the yeast Pichia pastoris has 402 large, clustered peroxisomes whereas small, diffused organelles are present when 403 grown in oleic acid (27). In another yeast, Hanensula polymorpha pexophagy is 404 induced when cells are moved from methanol to glucose media. 405
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We observed a change in glycosome composition when cells were passed back to 1 x 406 105/ml but not when they were passed to 5 x 105/ml. At this point we do not know 407 if this change in glycosome composition is the result of changes in cell density or 408 media composition as the cells were not washed between passages. It is possible 409 that the residual media carried with cells through passage affected the cellular 410 response. Separating these two variable has been difficult as washing the cells 411 before diluting them to 1 x105/ml greatly reduces cell viability. 412 413 In T. brucei, it is possible that the two parasite populations have different metabolic 414 capacities. Parasites containing dim glycosomes lacking aldolase and GAPDH may 415 rely heavily on amino acid metabolism and therefore do not require the large 416 number of glycolytic enzymes normally housed in the glycosome. 417 418 Additionally, de novo synthesized glycosomes may be primarily populated with 419 proteins carried from the ER, likely the source of origin. The lack of PEX13 may 420 block the import of metaboically important proteins and serve as a means to 421 regulate maturity. Further work on the protein composition and metabolic status of 422 each population will be required to address this model. 423 424 We do not know if glycosomes of BSF parasites exhibit the type of plasticity 425 observed in PF parasites. We predict that the obligate dependence on glucose 426 metabolism may prevent or repress dramatic changes in glycosome matrix protein 427 composition. 428
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Acknowledgements: We would like to acknowledge Wandy Beatty at the 429 Washington University School of Medicine, Medical Microbiology imaging facility (St. 430 Louis) for the TEM images. The work was supported in part by NIH 2R15A1075326 431 to JCM. 432 433
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