Understanding the interplay between intracellular pathogens and their hosts is vital to the development of new treatments and preventive strategies. The intracellular “amastigote” stage of the Chagas disease parasite, Trypanosoma cruzi , is a critical but understudied parasitic life stage.

We describe a system for the analysis of an important unicellular eukaryotic flagellate in a confining and crowded environment. The parasite Trypanosoma brucei is arguably one of the most versatile microswimmers known. It has unique properties as a single microswimmer and shows remarkable adaptations (not only in motility, but prominently so), to its environment during a complex developmental cycle involving two different hosts. Specific life cycle stages show fascinating collective behaviour, as millions of cells can be forced to move together in extreme confinement. Our goal is to examine such motile behaviour directly in the context of the relevant environments. Therefore, for the first time, we analyse the motility behaviour of trypanosomes directly in a widely used assay, which aims to evaluate the parasites behaviour in collectives, in response to as yet unknown parameters. In a step towards understanding whether, or what type of, swarming behaviour of trypanosomes exists, we customised the assay for quantitative tracking analysis of motile behaviour on the single-cell level. We show that the migration speed of cell groups does not directly depend on single-cell velocity and that the system remains to be simplified further, before hypotheses about collective motility can be advanced. Graphic abstract

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27Glycosomes are peroxisome-related organelles that compartmentalise the glycolytic enzymes 28 in kinetoplastid parasites. These organelles are developmentally regulated in their number 29 and composition, allowing metabolic adaptation to the parasite's needs in the blood of 30 mammalian hosts or within their arthropod vector. A protein phosphatase cascade regulates 31 differentiation between parasite developmental forms, comprising a tyrosine phosphatase, 32TbPTP1, that dephosphorylates and inhibits a serine threonine phosphatase TbPIP39 that 33 promotes differentiation. When TbPTP1 is inactivated, TbPIP39 is activated and during 34 differentiation becomes located in glycosomes. Here we have tracked TbPIP39 recruitment to 35 glycosomes during differentiation from bloodstream stumpy forms to procyclic forms. 36Detailed microscopy and live cell imaging during the synchronous transition between life 37 cycle stages revealed that in stumpy forms, TbPIP39 is located at a periflagellar pocket site 38 closely associated with TbVAP, that defines the flagellar pocket endoplasmic reticulum. 39 TbPTP1 is also located at the same site in stumpy forms, as is REG9.1, a regulator of 40 stumpy-enriched mRNAs. This site provides a molecular node for the interaction between 41 TbPTP1 and TbPIP39. Within 30 minutes of the initiation of differentiation TbPIP39 42 relocates to glycosomes whereas TbPTP1 disperses to the cytosol. Overall, the study 43 identifies a 'stumpy regulatory nexus' (STuRN) that co-ordinates the molecular components 44 of life cycle signalling and glycosomal development during transmission of Trypanosoma 45 brucei. 46 47 48 49 3 50 Importance 51 52 African trypanosomes are parasites of sub-Saharan Africa responsible for both human and 53 animal disease. The parasites are transmitted by tsetse flies and completion of their life cycle 54 involves progression through several development steps. The initiation of differentiation 55between blood and tsetse forms is signalled by a phosphatase cascade, ultimately trafficked 56 into peroxisome-related organelles called glycosomes that are unique to this group of 57 organisms. Glycosomes undergo substantial remodelling of their composition and function 58 during the differentiation step but how this is regulated is not understood. Here we identify a 59 cytological site where the signalling molecules controlling differentiation converge before the 60 dispersal of one of them into glycosomes. This coincides with a specialised ER site that may 61 contribute to glycosome developmental biogenesis or regeneration. In combination, the study 62 provides the first insight into the spatial co-ordination of signalling pathway components in 63 trypanosomes as they undergo cell-type differentiation. 64 65 66 4 67 Introduction 68The dynamic regulation of organelle biogenesis or composition often involves intimate 69 contact between the endoplasmic reticulum and the membrane of the target organelle (1). 70This enables the maturation and modification of organellar protein content, influ...