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Jul 15, 2023Nature Communications 13권, 기사 번호: 5326(2022) 이 기사 인용
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인간과 가축의 주요 병원체를 포함하는 트리파노소마티드(Trypanosomatids)는 편모가 있는 원생동물로 세포 주기 조절과 기본 메커니즘이 완전히 이해되지 않습니다. 여기에서는 Trypanosoma brucei의 세포 주기 결함에 대한 게놈 전체 RNA 간섭 라이브러리 화면을 설명합니다. 우리는 대규모 병렬 녹다운을 유도하고, 높은 처리량의 유동 세포 계측법, 각 단계의 심층 서열 RNAi 표적 및 게놈 규모에서 디지털 방식으로 재구성된 세포 주기 프로파일을 사용하여 교란된 인구를 분류했습니다. 또한 온라인 도구(https://tryp-cycle.pages.dev/)를 사용하여 데이터 시각화를 활성화합니다. 세포 주기 진행에 영향을 미치는 수백 개의 유전자를 분석하면 게놈 내재복제와 관련된 100개 이상의 편모 구성 요소 녹다운, G1-S 전이의 대사 조절에 대한 증거, G2M 축적과 연결된 표면 항원 조절 mRNA 결합 단백질 녹다운 및 추정되는 뉴클레오레독신이 필요함을 알 수 있습니다. 미토콘드리아 게놈 분리와 유사분열 모두에 대해. 결과는 트리파노소마 세포 주기 진행을 조정하는 알려지고 새로운 기계, 경로 및 조절기에 대한 포괄적인 기능적 게놈 증거를 제공합니다.
표준 진핵 세포 주기는 별개의 단계를 포함합니다: 세포가 DNA 복제를 준비하는 G1(갭 1); 핵 DNA 복제가 일어나는 S(합성) 단계; G2(갭 2): 세포가 유사분열을 준비할 때; 복제된 DNA가 분리되고 핵이 분열되는 경우 M(유사분열)1. 유사분열 후에는 세포질 분열(세포 분열)이 일어나 두 개의 딸세포가 생성됩니다2. 품질 관리를 용이하게 하는 속도 제한 메커니즘은 개별 지점에서 완화됩니다. 따라서 세포 주기가 진행되는 동안 발생하는 이상 현상은 세포 주기가 지연되거나 정지되어 세포가 이상 현상을 해결할 수 있도록 할 수 있습니다. 세포 사멸, 변칙이 해결될 수 없는 경우, 또는 다른 결과 중에서 발암의 경우. 따라서 세포주기를 통한 진행은 일반적으로 엄격한 체크포인트 제어하에 있습니다. G1-S, 내부 S 단계, G2-M 및 스핀들 체크포인트는 각각 S 단계의 시작, S 단계 진행, M 단계 및 M 단계 진행의 시작을 제어합니다3. 이러한 과정은 특히 세포주기 결함이 발암의 일반적인 유발 요인이기 때문에 광범위하게 연구되었습니다4. 그러나 진핵세포 주기 진행 제어의 진화와 메커니즘에 대한 우리의 이해는 주로 후편핵생물(동물과 균류 포함)에 대한 연구에서 비롯되었으며, 트리파노소마티드와 같은 다양한 진핵생물에 대한 연구는 상대적으로 적습니다.
트리파노소마티드는 편모가 있는 원생동물이며 인간과 동물의 건강에 큰 영향을 미치는 다양한 소외 열대 질병을 일으키는 기생충을 포함합니다. 아프리카의 트리파노소마(Trypanosoma brucei)는 체체파리에 의해 전염되며 사하라 사막 이남 아프리카 전역에서 인간과 동물의 질병, 수면병, 나가나를 각각 유발합니다5. T. brucei는 기생충과 모델 유기체로서 매우 다루기 쉬운 실험 시스템으로 나타났습니다6. 예를 들어, T. brucei 편모7는 인간 섬모병증8,9,10,11에 대한 연구를 위한 모델 역할을 합니다. Trypanosoma cruzi 및 Leishmania와 같은 다른 병원성 trypanosomatids와 공유되는 다양한 특징에는 글리코솜 내에서 구획화된 해당작용, 운동질체로 알려진 복잡한 미토콘드리아 DNA 구조를 가진 단일 미토콘드리아 및 거의 모든 유전자의 폴리시스트론 전사가 포함됩니다. 트리파노소마티드의 광범위하고 구성적인 폴리시스트론 전사는 예를 들어 mRNA 결합 단백질(RBP)에 의한 전사 후 조절과 단백질 인산화와 관련된 번역 후 조절에 중점을 두고 있습니다.
T. brucei의 세포 주기 제어에 초점을 맞춘 연구에서는 잘 연구된 다른 진핵생물에서 보존된 특징뿐만 아니라 다양한 특징도 밝혀졌습니다13,15. 특히, 이용 가능한 증거는 특정 세포주기 체크포인트가 없다는 것을 시사합니다. 예를 들어 T. brucei16의 곤충 단계에서는 세포질 분열이 유사분열이나 핵 DNA 합성과 무관하게 발생할 수 있습니다. 더욱이, 이전에는 고도로 보존된 단백질에 의해 충족된다고 생각되었던 기능은 트리파노소마티드에서 계통 특이적이거나 매우 다양한 단백질을 사용합니다. 염색체 분리를 지시하는 동원체 복합체는 예를 들어 트리파노소마티드에 특이적인 반면, DNA 복제 개시에 관여하는 기원 인식 복합체(ORC)는 매우 다양합니다. 높은 처리량 연구 측면에서 T. brucei 세포 주기 동안 전사체19 및 프로테옴20 모니터링을 통해 수백 개의 조절된 mRNA 및 단백질이 밝혀졌고, 인단백질 분석에서는 여러 RBP21의 동적 인산화가 밝혀졌습니다. 그러나 많은 T. brucei 유전자가 아직 특정 기능을 지정하지 않았고 많은 세포 주기 조절인자가 확인되지 않은 상태로 남아 있기 때문에 분기는 상당히 뛰어난 과제를 제시합니다. 높은 처리량, 게놈 규모의 기능적 유전 스크린을 사용하여 특정 과정에서의 역할에 대해 게놈의 모든 유전자를 동시에 평가할 수 있습니다. 우리는 T. brucei22에 대한 RNA 간섭 표적 시퀀싱(RIT-seq)을 개발했으며 이전에 생성된 게놈 규모 피트니스 프로파일을 통해 필수성 예측과 잠재적인 약물 표적의 우선순위 지정을 촉진했습니다.
4 C) cells (Fig. 1a). Polyploid cells arise due to endoreduplication, additional rounds of DNA replication without cytokinesis, either with24 or without25,26 mitosis, yielding cells with multiple nuclei or with polyploid nuclei, respectively./p>4 C (multiple nuclei) phenotypes; anuclear zoids are not shown as they are undetectable by RIT-seq. b The schematic illustrates the RIT-seq screen; massive parallel induction of RNAi with tetracycline (Tet), followed by flow cytometry and RIT-seq, allowing for reconstruction of cell cycle profiles, using mapped reads from each knockdown. Each read-mapping profile encompasses the gene of interest and associated untranslated regions present in the cognate mRNA. The library data represents the uninduced and unsorted population. GeneIDs, Tb927.7.3160 for example, are indicated without the common ‘Tb927.’ component./p>4 C) pools (Supplementary Fig. 1). Fixation and staining with the fluorescent DNA intercalating dye were pre-optimised for high-throughput sorting (see Materials and Methods). Approximately 10 million cells were collected for each of the G1, S and G2M pools and samples from these pools were checked post-sorting to assess their purity (Fig. 1b, Supplementary Fig. 1). For the perturbed and less abundant <2 C and >4 C pools, less than one million cells were collected; these pools were retained in their entirety for RIT-seq analysis./p>7200 non-redundant gene sequences in the uninduced and induced, unsorted library controls and in each of the five sorted samples. We selected the 24 h timepoint, equivalent to approximately 3.5 population doubling times, for the current analysis. We found that reads for 23.4% of genes were diminished by >3-fold following 72 h of knockdown in our prior RIT-seq study23, while reads for only 0.6% of genes dropped by >3-fold following 24 h of knockdown in the unsorted control samples analysed here (see Supplementary Fig. 2a, b). Thus, 24 h should have allowed sufficient time for the development of robust inducible phenotypes and also captured perturbed cells before they were critically diminished due to loss-of-fitness. An unanticipated feature that emerged from this analysis of prior RIT-seq data was that knockdown of proteins associated with DNA replication typically failed to register a major loss-of-fitness (Supplementary Fig. 2a, b). This suggested that a reduced rate of DNA replication can be tolerated, albeit extending S phase (see below) but having relatively little impact on viability. Each sorted sample library yielded between 23 and 37 million mapped read-pairs; <2 C = 37 M, G1 = 35 M, S = 30 M, G2M = 23 M, > 4 C = 25 M; this set of five samples yielded data for >7000 genes which equates to >35,000 RNAi data-points (Supplementary data 1)./p>4 C overrepresented, following knockdown (Fig. 1b). These outputs suggest that loss of a cytoplasmic dynein heavy chain (7.3160) does not perturb cell cycle distribution; that the proteasome is required to complete G2M (see below); and that knockdown of a flagellar axonemal dynein heavy chain (11.11220) results in endoreduplication in the absence of cytokinesis; dyneins are cytoskeletal motor proteins that either move along microtubules or drive microtubule sliding, to produce a flagellar beat, for example30./p>4 C cells, indicating endoreduplication, which yielded 284 genes (Fig. 2a, left-hand panel; Supplementary data 1). The >4 C phenotype was previously observed following α-tubulin knockdown in a landmark study that first described RNAi in T. brucei24 and, indeed, we observed pronounced overrepresentation of >4 C cells for both adjacent α-tubulin and β-tubulin gene knockdowns (Fig. 2a, middle and right-hand panel). We then examined knockdowns reporting an overrepresentation of <2 C cells, indicating a reduced DNA content, which yielded 10 hits (Fig. 2b, left-hand panel; Supplementary data 1). Haploid cells were previously observed following DOT1A knockdown31 and, consistent with the previous report, we observed pronounced overrepresentation of <2 C cells for the DOT1A gene knockdown (Fig. 2b, middle and right-hand panel); we are not aware of other knockdowns reported to yield a similar phenotype. Indeed, other ‘<2 C hits’ mostly encode small hypothetical proteins, seven of which are 73 ± 11% shorter than the average, consistent with low read-count and under-sampling for these hits (Supplementary Fig. 2c). The remaining two hits are a histone chaperone (ASF1B) and a glycolytic enzyme (PFK). Together, these results provided initial validation for the >4 C and <2 C components of the screen./p>4 C experiment in red; those with reads in the >4 C pool that exceeded the mean fold-change value by >1.75 times the SD, equivalent to >1.117-fold the sum of reads in the G1, S phase and G2M samples combined. The read-mapping profile and read-counts for α/β-tubulin are shown to the right. b The plot on the left shows knockdowns overrepresented in the sub-2C experiment in orange; those with reads in the sub-2C pool that exceeded the mean fold-change value by >1.75 times the SD, equivalent to >4-fold the sum of reads in the G1, S phase and G2M samples combined. The read-mapping profile and read-counts for DOT1A are shown to the right. c The RadViz plot shows knockdowns that registered >25% overrepresented read-counts in the G1 (purple), S phase (green), or G2M (blue) categories. d Read-mapping profiles and relative read-counts for example hits. PCNA, proliferating cell nuclear antigen; PPL2, PrimPol-like 2. e The Venn diagram shows the distribution of knockdowns overrepresented in each arm of the screen./p>25% overrepresented read counts in each of these categories are highlighted in the RadViz plot in Fig. 2c (also see Supplementary data 1) and data for an example from each category are shown in Fig. 2d; the glycolytic enzyme, aldolase, reported 104% increase in G1 cells (further details below); the proliferating cell nuclear antigen (PCNA), a DNA sliding clamp that is a central component of the replication machinery32, reported 25% increase in S phase cells and 13% increase in G2M cells, consistent with prior analysis33; and PrimPol-like 2 (PPL2), a post-replication translesion polymerase, reported 65% increase in G2M cells, also consistent with prior analysis34. These results provided initial validation for the G1, S phase and G2M components of the screen. The full dataset can be searched and browsed using an interactive, open access, online data visualization tool (see Supplementary Fig. 3; https://tryp-cycle.pages.dev/)./p>4 C, p = 9.4−213), consistent with loss-of-fitness as a common outcome following a cell cycle progression defect. Taken together, the analyses above provided validation for the RIT-seq based cell cycle phenotyping approach and yielded >1000 candidate proteins that impact progression through specific steps of the T. brucei cell cycle./p>4 C cells can arise due to endoreduplication without cytokinesis, either with24 or without25,26 mitosis. Endoreduplication defects were previously observed following knockdown of α-tubulin24 or flagellar proteins7,35; consistent with the view that flagellar beat is required for cytokinesis in bloodstream form T. brucei. As shown above, dynein heavy chain (see Fig. 1b), α-tubulin and β-tubulin (see Fig. 2a) knockdowns were amongst 284 knockdowns overrepresented in the endoreduplicated pool in our screen. Gene Ontology (GO) annotations, which provide structured descriptions of gene products in terms of functions, processes, and compartments, were assessed to further profile this cohort of knockdowns. Terms overrepresented in association with an endoreduplication defect included ‘dynein’, ‘intraflagellar transport’ (IFT), ‘axoneme’ and ‘cytoskeleton’, and also ‘chaperonin T-complex’, ‘cytokinesis’ and ‘cell cycle’ (Fig. 3a). The violin plot in Fig. 3b shows specific enrichment of IFT and dynein knockdowns in association with endoreduplication. Exocyst components, primarily involved in exocytosis36, were included as a control cohort since none of the exocyst components registered enrichment in the >4 C pool, nor in any other experimental pool analysed here (see below). Enrichment of individual chaperonin T-complex components, dyneins, and IFT factors in the >4 C pool is illustrated in Fig. 3c. The chaperonin T-complex is involved in tubulin and actin folding37 and, notably, actin knockdown was also associated with endoreduplication (Supplementary Fig. 4)./p>4 C hits, those that exceed the mean fold-change value in this set by >1.75 times the SD. P-values are shown on the right. b The violin plot shows relative >4 C read-counts for cohorts of genes and reflects data distribution. Open circles indicate median values and the vertical bars indicate 95% confidence intervals. Significantly overrepresented cohorts are indicated in red. c The plots show overrepresentation of T-complex, dynein and intraflagellar transport (IFT) factors in red in the >4 C experiment. d The heatmaps show relative representation in all five sorted pools for the above and additional cohorts of knockdowns; blue, most overrepresented. e Example read-mapping profiles for hits overrepresented in the >4 C pool. f Example read-mapping profiles for ciliopathy-associated hits overrepresented in the >4 C pool. CMF Component of Motile Flagella, CFAP Cilia and Flagella Associated Protein./p>4 C pool; these include additional dynein chains, radial spoke proteins, extra-axonemal paraflagellar rod (PFR) proteins, as well as nucleoporins. The gallery in Fig. 3e shows examples of RIT-seq read-mapping profiles for twenty-six individual genes that register >4 C enrichment following knockdown. In addition to the categories above, these include the inner arm dynein 5-138, FAZ proteins which mediate attachment of the flagellum to the cell body39; all four cytokinesis initiation factors CIF1-440, and chromosomal passenger complex components, including CPC1 and the aurora B kinase, AUK1. AUK1 and CPC1 are spindle-associated and regulate mitosis and cytokinesis26,41. Notably, endoreduplication was reported previously following AUK1 knockdown in bloodstream form T. brucei42 and this is the kinase with the most pronounced overrepresentation in our >4 C dataset. The next >4 C overrepresented kinase is the CMGC/RCK (Tb927.3.690), knockdown of which previously yielded a striking cytokinesis defect43./p>4 C overrepresentation include the centriole cartwheel protein SAS644, the cleavage furrow-localizing protein FRW145, the basal body—axoneme transition zone protein TZP12546 and the basal body protein BBP24847. One hundred additional examples are shown in Supplementary Fig. 4, including intermediate and light chain dyneins, other flagellum-associated factors, radial spoke proteins, components of motile flagella, flagellum attachment and transition zone proteins, kinesins48,49, nucleoporins50, and many previously uncharacterised hypothetical proteins. Some other notable examples include the microtubule-severing katanin KAT8051, the dynein regulatory factor trypanin52, the AIR9 microtubule associated protein53, CAP51V54 and importin, IMP155./p>4 C pool are shown in Fig. 3f and Supplementary Fig. 4. These include orthologues of proteins linked to primary ciliary dyskinesia (DNAH5, DNAH11, RSPH4 and DNAI1)11; male infertility (PF16, PACRGA, CFAP43 and CMF7/TbCFAP44)7,10; and cone-rod dystrophies, as well as other ocular defects (CMF17, CMF39 and CMF46)8./p>4 C pool, we conclude that RIT-seq screening provided comprehensive genome-scale identification of cytokinesis defects in bloodstream form T. brucei. Endoreduplication appears to be a common outcome following a cytokinesis defect. Amongst hundreds of genes required for progression through cytokinesis, flagellar proteins featured prominently, including the majority of dynein chains and intraflagellar transport factors. Many of these factors are essential for viability and include potential druggable targets in trypanosomatids, as well as orthologues of proteins associated with ciliopathies./p>25% overrepresented read counts in each of these categories (Fig. 2c, e). GO annotations within each cohort revealed a number of enriched terms (Fig. 4a). Overrepresented knockdowns were associated with glycolysis, mRNA binding and the mitochondrion in the G1 pool, with DNA replication in the S phase pool and with a broadly similar profile to that seen for the >4 C set in the G2M pool./p>25% overrepresented read-count in each of these categories. P-values are shown on the right. b The violin plots show relative G1, S phase or G2M read-counts for cohorts of genes and reflect data distribution. Open circles indicate median values and the vertical bars indicate 95% confidence intervals. Overrepresented cohorts are indicated in purple, green and blue, respectively. IFT intraflagellar transport. c The heatmaps show relative representation in all five sorted pools for the above and additional cohorts of knockdowns; blue, most overrepresented. MCM minichromosome maintenance, PSP1 DNA polymerase suppressor 1./p>4 C pools likely reflects cytokinesis defects with cells accumulating both before and after endoreduplication; compare G2M and >4 C data for IFT factors and dyneins in Fig. 4b and Fig. 3b, for example. Other mitosis or cytokinesis-perturbed phenotypes are likely not associated with substantial endoreduplication; see the kinetochore and proteasome cohorts in Fig. 4b, for example. Once again, the exocyst provided a control cohort with no components registering enrichment in the G1, S phase or G2M pools following knockdown (Fig. 4b)./p>25% overrepresentation in the G1 pool; hexokinase, phosphofructokinase, aldolase (see Fig. 2c), triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, phosphoglycerate kinase C and pyruvate kinase. Glycolysis operates in peroxisome-like organelles known as glycosomes in trypanosomes and is thought to be the single source of ATP in bloodstream form cells12. Glycolysis also provides metabolic intermediates that support nucleotide production. Notably, mammalian cell proliferation is accompanied by activation of glycolysis, and the Warburg effect relates to this phenomenon in oncology60,61. Indeed, hexokinase regulates the G1/S checkpoint in tumour cells62. The results are also consistent with the observation that T. brucei accumulate in G1 or G0 under growth-limiting conditions63 or during differentiation to the non-dividing stumpy form64, possibly reflecting a role for glucose sensing in differentiation65. Notably, glycolytic enzymes are downregulated 6.7 + /−5.2-fold in stumpy-form cells66. We conclude that, as in other organisms67, there is metabolic control of the cell cycle and a nutrient sensitive restriction point in T. brucei, with glycolysis playing a role in the G1 to S phase transition and possibly also the G1/G0 transition./p>25% overrepresented read-counts in the G1 category are indicated. Black data-points indicate other genes from each cohort. Grey data-points indicate all other genes. The read-mapping profiles and relative read-counts in the lower panel show example hits. b As in a but for DNA replication initiation factor knockdowns that registered >25% overrepresented read-counts, primarily in the S phase category. c As in a but for proteasome component knockdowns that registered >25% overrepresented read-counts, primarily in the G2M category. d As in a but for kinetochore component knockdowns that registered >25% overrepresented read-counts, primarily in the G2M category./p>25% overrepresentation in the S phase pool are components of the eukaryotic replicative helicase, the CMG (Cdc45-MCM-GINS) complex. At the core of this complex is the minichromosome maintenance complex (MCM2-7), a helicase that unwinds the duplex DNA ahead of the moving replication fork68. Identification of CMG complex components suggests that each of these subunits is required for timely progression through S phase./p>25% overrepresentation in the G2M pool. This output is consistent with the view that the T. brucei proteasome is responsible for degrading cell cycle regulators, such as poly-ubiquitinated cyclins, some of which are known to control cell cycle checkpoints in T. brucei. Candidate targets in T. brucei include: CIF1, AUK170, cyclin 6 (CYC6), degradation of which is required for mitosis71; cyclin-like CFB2, required for cytokinesis72; and cyclin 2 (CYC2) or cyclin 3 (CYC3), which have short half-lives and a candidate destruction box motif in the case of CYC373./p>25% overrepresentation in the G2M pool, suggesting that these particular kinetochore components, which all display temporal patterns of phosphorylation from S phase to G2M21, are required for progression through mitosis. Notably, KKT10 is a kinase responsible for phosphorylation of KKT7, which is required for the metaphase to anaphase transition74; as well as for the phosphorylation of KKT1 and KKT2, in turn required for kinetochore assembly75,76. These findings are consistent with the view that kinetochore components control a non-canonical spindle checkpoint in trypanosomes74./p>25% overrepresentation in these pools (Fig. 6a). These include knockdowns for RBP10 and RBP29 enriched in G1; RBP10, in particular, has been characterised in some detail and promotes the bloodstream form state77. ZC3H1178, ZC3H41 and ZC3H2879 knockdowns were enriched in G1, S phase and G2M, respectively, while knockdowns of CFB2, MKT1 or PBP1, all recently linked to variant surface glycoprotein expression control80,81, were enriched in G2M. Indeed, based on the outputs of the current screen, we prioritised these latter three RBPs for follow-up analysis in a separate study; all three were thereby validated as G2M hits80. Thus, the RIT-seq cell cycle screen implicated a number of specific RBPs in post-transcriptional control of cell cycle progression through modulation of mRNA stability and/or translation./p>25% overrepresented read-counts in the G1, S phase or G2M categories are indicated, in purple, green and blue, respectively. The read-mapping profiles and relative read-counts in the other panels show example hits. b As in a but for protein kinase knockdowns, with selected kinases indicated. c As in a but for hypothetical (conserved) protein knockdowns./p>4 C (Fig. 3d), S phase or G2M (Fig. 5d) phenotypes, and now show the RIT-seq profiling data for five additional protein kinase knockdowns that register >25% overrepresentation in the G1, S phase or G2M pools (Fig. 6b). These include knockdowns for CRK7, linked to accumulation in G1; MAPK5, linked to accumulation in S phase and polo-like kinase (PLK) and cdc2-related kinase 3 (CRK3), linked to accumulation in G2M. PLK was previously shown to control cell morphology, furrow ingression and cytokinesis82,83,84, while CRK3 was shown to play a role in G2M progression in bloodstream form T. brucei43,85. Overall correspondence was also excellent with a prior kinome-wide RNAi screen43. For example, eight among nine kinases linked to a mitosis defect in that screen also reported an (21 ± 12%) increase in the G2M pool in the current screen./p>2500 genes. We show data for several hypothetical protein knockdowns above, linked to the enriched >4 C phenotype (Supplementary Fig. 4), and we here identify >300 additional hypothetical protein knockdowns that register >25% overrepresentation in the G1, S phase or G2M pools. RIT-seq profiling data are shown for five examples in Fig. 6c and for several additional examples in Supplementary Fig. 5. Amongst other examples of knockdowns shown in Supplementary Fig. 5, are alternative oxidase86, linked to G1 enrichment; kinesins linked to G2M enrichment, including both chromosomal passenger complex kinesins (KIN-A and KIN-B)26 and KIN-G; CYC625,87, centrin 388 and, finally, both components of the histone chaperone FACT (facilitates chromatin transcription) complex89 Spt16 and Pob3, linked to G2M enrichment. Notably, the FACT complex has been linked to centromere function in human cells90./p>4 C pools (χ2 p = 8.9−9) following knockdown, including kinetochore and chromosomal passenger complex components, respectively. Some specific transcripts required for cell cycle progression may be upregulated prior to peak demand for the encoded protein, and we found evidence to support this view. For example, transcripts upregulated in late G1 or in S phase were enriched amongst those knockdowns linked to accumulation in the G2M pool (χ2 p = 3.3−3 and p = 0.011, respectively); both components of the FACT complex, upregulated in G1, for example (see Supplementary Fig. 5). Similarly, S phase and G2M upregulated transcripts, including those encoding multiple flagellum-associated proteins, were enriched amongst knockdowns linked to accumulation in the >4 C pool (χ2 p = 4.6−18 and p = 2.4−5 respectively)./p>4 C hit in bloodstream-form trypanosomes, we assembled a pair of independent inducible RNAi knockdown strains. Analysis of cell growth revealed a severe loss-of-fitness following knockdown, confirmed by qRT-PCR (Fig. 7c). Flow cytometry then confirmed endoreduplication, with prominent peaks detected representing 8 C and 16 C cells following knockdown (Fig. 7d, left-hand panel), while examination of these cells by microscopy revealed multiple nuclei, indicating endoreduplication with continued mitosis (Fig. 7d, right-hand panel)./p>4 C (~9 × 105 cells) based on their DNA content and collected into 50 ml Falcon tubes (BD Falcon); total sorting time was approx. 4 h. The 2 C, 2–4 C and 4 C sorted samples were then run on a FACS LSR Fortessa flow cytometry analyser for a post-sorting quality check. For the analysis of Tb927.10.970 or Tb927.10.3970 knockdowns, 1 × 107 cells were centrifuged for 10 min at 1000 g, washed in supplemented PBS. Cells were fixed for 10 min in 1% paraformaldehyde in supplemented PBS, washed and stored at 4 °C in supplemented PBS. Cells were pelleted for 10 min at 1000 g and permeabilised at room temperature for 30 min in supplemented PBS plus 0.01% Triton X-100. Cells were washed once in supplemented PBS followed by centrifugation for 10 min at 700 g and then stained in supplemented PBS with 10 μg.ml−1 propidium iodide and 100 μg.ml−1 RNAse A for 1 h at 37 °C. The samples were run on a BD FACSCanto (Becton Dickinson). FlowJo v10.7.1 was used for data analysis and visualisation./p> 
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