TbMyo1 has a large cytosolic pool.

(A) Schematic representation of the two-step fractionation protocol. BSF T. brucei cells were mixed with GFP-expressing BSF cells and extracted with digitonin to release the cytosolic fraction. The permeabilised cells were subsequently extracted with IGEPAL (non-ionic detergent) to release the organelle fraction. 5% samples of the input (I), supernatant (SN1, SN2) and pellet (P1, P2) fractions were taken at the indicated points. The samples were separated by SDS-PAGE and analysed by immunoblotting. (B) TbMyo1 is present in both a cytosolic and a cytoskeleton-associated pool. Exemplary immunoblotting results from the two-step fractionation protocol as described in (A). Equal fractions (∼2%) were loaded in each lane. The cytosolic marker (GFP, ∼ 27 kDa) was mainly detected in the SN1 fraction. The ER chaperone BiP (∼ 80 kDa) partitioned into the P1 and then into the SN2 fractions. The flagellar cytoskeleton proteins PFR1,2 (∼ 79 + 82 kDa) partitioned into the P1 and then the P2 fractions. TbMyo1 (∼ 130 kDa) was found to be nearly equally divided between the SN1 and P1 fractions. The P1 fraction almost completely partitioned into the P2 fraction. Exemplary images from multiple (n = 8) independent experiments are shown. L, molecular weight ladder. (C) Nearly 50% of TbMyo1 is cytosolic. Quantification of the fractionation immunoblot data (n = 8). The red bars indicate the median.

TbMyo1 in vitro translocates actin in vitro.

(A) Schematic illustration and structural prediction of the domain architecture of TbMyo1. The motor domain is shown in cyan, the IQ (calmodulin-binding) motif is shown in yellow, the FYVE domain is shown in magenta. Regions without domain prediction are displayed in grey. The structural prediction was generated using AlphaFold (ID: AF-Q585L2-F1). (B) Size exclusion profile of affinity-purified TbMyo1 (Superdex 200 Increase 10/300). The monomeric TbMyo1 fraction eluted in a single peak at around 12 ml. The 9 ml (void volume) elution peak corresponds to aggregated protein.

(C) TbMyo1 eluted mainly in the second protein peak. Samples were taken from the eluted volumes corresponding to the different protein peaks and analysed by SDS-PAGE. TbMyo1 (130 kDa) eluted together with human Calmodulin in the 11.5 ml to 12.5 ml elution fractions.

(D) Schematic representation of the in vitro actin filament gliding assay. Monomeric, C-terminally biotinylated TbMyo1 was immobilised via streptavidin on a glass surface. TbMyo1 translocated Alexa488-phalloidin labelled filamentous actin (F-actin) in the presence of ATP.

(E) TbMyo1 translocates filamentous actin in vitro. Measurements were taken at 22°C with a final concentration of 2 mM ATP (n = 30 actin filaments). The average velocity was 130 ± 40 nm/s (mean ± SD; Gaussian fit).

TbMyo1 is associated with the endocytic and not the biosynthetic pathway.

(A) TbMyo1 is concentrated on structures in the posterior region of the cell. Expansion microscopy of bloodstream form (BSF) T. brucei labelled with anti-tubulin (yellow), anti-TbMyo1 (magenta) and DAPI (cyan). Six exemplary cells are shown (I – VI). For all cells, a magnified image of the posterior region (boxed area) is shown without the tubulin signal. The TbMyo1 signal was visible as a large cluster of foci in the posterior region between nucleus (N) and kinetoplast (K). (B) TbMyo1 does not colocalise with the ER (BiP, panels I and II), Golgi apparatus (GRASP, panels III and IV), or lysosome (p67, panels V and VI) markers. Fixed BSF cells were labelled using the indicated antibodies or tags and imaged using widefield microscopy and structured illumination microscopy (SIM). Overlay images with DIC and DAPI are shown for widefield microscopy images; SIM images show a merge of all three fluorescence channels. Magnified images of the regions of interest for TbMyo1 and the indicated markers are shown. (C) Quantification of correlation between TbMyo1 and organelle marker signals. Correlation was estimated using Spearman’s rank correlation (BiP/TbMyo1 n = 38, GRASP/TbMyo1 n = 28, p67/TbMyo1 n = 27). The median ρ is represented by a red line and the corresponding number is written below the data plot. (D) TbMyo1 overlaps with endocytic cargo. Cells expressing mNG-TbMyo1 were incubated with fluorescent cargo, fixed, and imaged using widefield microscopy. TbMyo1 (green) and the cargo markers (magenta) showed partial (BSA, panel I) or strong (transferrin, Tf, panel II) overlap. A magnified image of the fluorescence channels and a merge are shown next to the overlay with DIC. (E) Quantification of correlation between TbMyo1 and endocytic cargo marker signals. Correlation was estimated using Spearman’s rank correlation (BSA/ TbMyo1 n = 69, Tf/ TbMyo1 n = 77). The median ρ is represented by a red line and the corresponding number is written below the data plot. Exemplary images from multiple (n > 2) independent experiments are shown.

Ultrastructural localisation of TbMyo1.

(A - H) TbMyo1 localises to endosomal tubes, vesicles, glycosomes, the flagellar pocket, and the cytoplasm. Electron micrographs of cryosections labelled with anti-TbMyo1 and 12 nm gold-conjugated secondary antibodies. For each panel a raw and a pseudocoloured version of the image are presented. Endosomal tubes are coloured green. Vesicles are coloured blue. Glycosomes are outlined in yellow. The lysosome is coloured brown. Gold particles corresponding to TbMyo1 signals are outlined in magenta. (F - H) Cells were incubated with BSA conjugated to 5 nm gold particles prior to fixation and immunolabelling with anti-TbMyo1. The BSA was observed in vesicles, endosomes, and the lysosome. (I) Distribution of TbMyo1 in the cytoplasm and on subcellular structures. All gold particles 30 nm or closer to a structure were defined as being on the structure (based on a conservative calculation that two antibodies, each 15 nm in length, are between the bound epitope and the gold particle). More than half of the gold particles were found to be cytoplasmic. The total number of cells and gold particles quantified is indicated. Abbreviations: axoneme (Ax), flagellum (F), flagellar pocket (FP), plasma membrane (PM), nucleus (N). Exemplary single- and double-labelled images were chosen from three separate labelling experiments.

TbMyo1 is clustered adjacent to the lysosome.

(A - D) Correlative light and electron microscopy (CLEM) imaging of cryosections. To visualise cargo trafficking, cells were incubated with double-labelled Alexa555- and 5 nm gold-conjugated BSA (cyan) before fixation. Cryosections were labelled with anti-TbMyo1 (magenta), anti-VSG (yellow), and DAPI (grey). For each panel an image of the widefield microscopy (I), the electron micrograph (II) and an overlay of both images (III) is presented. The electron micrographs are manually-stitched mosaics composed of multiple (4 – 9) tiled images of higher magnification (15,000x – 25,000x). Note that a fluorescence signal could only be detected at high local concentrations of Alexa555-BSA-5 nm gold. VSG and DAPI signals were used as fiducials to correlate IF and EM images. Correlations were made using the ec-CLEM plug-in in Icy for the initial correlation and Adobe Photoshop for the final overlay. Abbreviations: L – lysosome, * - glycosomes; the arrow in panel B shows endosomal tubes filled with BSA gold and positive for TbMyo1. Images show exemplary images from a sample of 17 correlated cells.

TbMyo1 overlaps extensively but not completely with actin.

(A) Imaging of trypanosome actin using an inducibly-expressed anti-actin chromobody. Tetracycline (Tet) was used to induce expression of the anti-actin chromobody. Widefield microscopy of fixed cells in the absence (- Tet) or presence (+ Tet) of tetracycline are shown. The induced (+ Tet) cells showed a chromobody signal (magenta) in the posterior region of the cell between the nucleus (N) and kinetoplast (K), consistent with the reported localisation of actin in BSF T. brucei. DNA was stained with DAPI. (B) Cell-to-cell variation of the chromobody signal in the tetracycline-induced population. The intensity but not the distribution of the signal varied from cell to cell. (C) The actin depolymerising drug latrunculin A (LatA) disrupts the chromobody signal. Widefield microscopy of fixed, induced chromobody cells in the presence of different LatA concentrations. The cells in the absence of LatA (- LatA) acted as controls. The addition of 0.2 μM LatA led to a slight blurring of the chromobody signal (magenta), while 2 μM LatA resulted in strong blurring (I) or even loss of signal (II) accompanied by a disruption of cell morphology. (D) TbMyo1 strongly overlaps with the anti-actin chromobody signal. Non-induced cells acted as a control and showed no chromobody signal, with only a TbMyo1 signal (green). Induced cells displayed both a chromobody (magenta) and a TbMyo1 signal, which strongly overlapped in the posterior region of the cell. (E) Quantification of correlation between TbMyo1 and actin signals. Correlation was estimated using Spearman’s rank correlation (n = 23). The median ρ is represented by a red line and the corresponding number is written above the data plot. (F) Enlarged view of the strong overlap between TbMyo1 and anti-actin chromobodies in the posterior region of the cell. Magnified images of the posterior region of four different cells (I – IV) are shown. TbMyo1 and the anti-actin chromobodies strongly overlapped in all cells examined, but the number and intensity of discrete spots varied.

Actin depolymerisation disrupts the endosomal system.

(A) LatA treatment perturbs the endosomal system. EP1-GFP expressing cells were incubated with 2 μM LatA for the indicated time periods, then fixed and imaged using widefield microscopy. DNA was stained using DAPI. Longer incubation with LatA resulted in aberrant cell morphologies and an increasingly diffuse EP1-GFP signal. Exemplary cells from a single experiment are shown. (B) Quantification of the morphological effects of LatA treatment. Cells were manually classified as displaying normal, swollen posterior, or rounded morphology. The degree of morphological disruption was proportional to the duration of incubation with LatA. Data acquired from >200 cells assayed in three independent experiments. (C) LatA treatment disrupts the ultrastructural morphology of the endosomal system. Cells expressing EP1-GFP were subjected to a 30 – 60 minute incubation with 2 μM LatA, followed by high-pressure freezing, embedding in Epon, and examination through transmission electron microscopy. The cytoplasm contained many large and interconnected tubular profiles, consistent with a swollen endosomal system. An enlargement of the flagellar pocket was additionally observed. Exemplary cells from a single experiment are shown.