Lysosome assembly and disassembly changes endocytosis rate through the Leishmania cell cycle

Abstract The Leishmania lysosome has an atypical structure, consisting of an elongated vesicle‐filled tubule running along the anterior–posterior axis of the cell, which is termed the multivesicular tubule (MVT) lysosome. Alongside, the MVT lysosome is one or more microtubules, the lysosomal microtubule(s). Previous work indicated there were cell cycle‐related changes in MVT lysosome organization; however, these only provided snapshots and did not connect the changes in the lysosomal microtubule(s) or lysosomal function. Using mNeonGreen tagged cysteine peptidase A and SPEF1 as markers of the MVT lysosome and lysosomal microtubule(s), we examined the dynamics of these structures through the cell cycle. Both the MVT lysosome and lysosomal microtubule(s) elongated at the beginning of the cell cycle before plateauing and then disassembling in late G2 before cytokinesis. Moreover, the endocytic rate in cells where the MVT lysosome and lysosomal microtubule(s) had disassembled was extremely low. The dynamic nature of the MVT lysosome and lysosomal microtubule(s) parallels that of the Trypanosoma cruzi cytostome/cytopharynx, which also has a similar membrane tubule structure with associated microtubules. As the cytostome/cytopharynx is an ancestral feature of the kinetoplastids, this suggests that the Leishmania MVT lysosome and lysosomal microtubule(s) are a reduced cytostome/cytopharynx‐like feature.


| INTRODUC TI ON
The kinetoplastids are flagellated single-celled eukaryotes whose shape and form are defined by a regular array of subpellicular microtubules. They have a single flagellum that extends from the flagellar pocket, which is an invagination of the cell body membrane and the site of the trafficking of macromolecular material into and out of these organisms (Field & Carrington, 2009). Many kinetoplastid parasites, including Leishmania and Trypanosoma cruzi, also have additional microtubules within the cytoplasm with one end of these microtubules normally positioned in close proximity to the flagellar pocket (Alcantara, Vidal, Souza, & Cunha-e-Silva, 2014;Lacomble et al., 2009;Wheeler, Sunter, & Gull, 2016). For kinetoplastid cell forms with the flagellum laterally attached to the side of the cell body, such as the Trypanosoma brucei trypomastigote, the subpellicular microtubule array is interrupted by a specialized set of four microtubules called the microtubule quartet (MtQ) that forms part of the flagellum attachment zone (Lacomble et al., 2009;. The flagellum attachment zone MtQ is nucleated close to the base of the flagellar pocket and then wraps around the pocket before invading the subpellicular array, following the line of flagellum attachment (Lacomble et al., 2009). In the Leishmania promastigote form, which does not have lateral attachment of the flagellum to the cell body, the flagellum attachment zone MtQ is present around the flagellar pocket and does not invade the subpellicular microtubule array (Wheeler et al., 2016).
The terminal endocytic compartment in Leishmania, the multivesicular tubule (MVT) lysosome, has a structure atypical of a lysosome. The MVT lysosome comprises a long vesicle-filled tubule that stretches from the flagellar pocket region at the cell anterior beyond the nucleus into the posterior end of the cell and is associated with one or two microtubules (Mullin et al., 2001;Waller & McConville, 2002;Weise, Stierhof, Kuhn, Wiese, & Overath, 2000). A low pH is important for the maintenance of this elongated tubular structure as the addition of bafilomycin A 1 , a specific inhibitor of vacuolar-type H + ATPases, caused a rapid collapse in the MVT lysosome (Mullin et al., 2001). The MVT lysosome contains cysteine and serine proteases as expected for a degradative compartment; however, the pH of this organelle appears less acidic than typical lysosomes as it is not readily stained with lysotracker, which accumulates in low pH organelles (Besteiro, Williams, Coombs, & Mottram, 2007;Mullin et al., 2001). The MVT lysosome was identified by Ilgoutz and colleagues using BODIPY-C 5 -Cer and a GFP tagged dolichol-phosphate-mannose synthase (DPMS) and was initially called the DPMS tubule (Ilgoutz, Mullin, Southwell, & McConville, 1999). Subsequent work by Weise and colleagues showed that this DPMS tubule was likely to be a lysosomal compartment; this was confirmed through further work by Mullin and colleagues who showed by immunoelectron microscopy that DPMS localized to the MVT lysosome (Mullin et al., 2001;Weise et al., 2000).
The lysosome in T. brucei does not have the elongated tubule structure observed in Leishmania and instead forms a rounded vesicular structure on the posterior side of the nucleus (Halliday et al., 2019;Langreth & Balber, 1975;Peck et al., 2008). The presence of a lysosome in T. cruzi has been the subject of debate: The terminal endocytic compartment was initially termed a reservosome as the structure lacked acid phosphatase activity and was not labeled with antibodies that recognize mammalian lysosome membrane proteins (Soares, Souto-Padrón, & Souza, 1992). Further work has shown that there are generally multiple reservosomes in a cell, which are spherical membrane-bound structures found in the posterior end of the cell with characteristics of prelysosomes, lysosomes, and recycling compartments, and have now been classified as lysosomal-related organelles (Cunha-e-Silva et al., 2006;Sant'Anna et al., 2008).
Trypanosoma cruzi has an additional endocytic organelle, the cytostome/cytopharynx, which is a long membrane tube that invades deep into the cell body with the entrance positioned close to the flagellar pocket. The cytostome/cytopharynx is the major route for bulk endocytosis into this parasite, and this structure is not found in Leishmania and T. brucei, but was likely present in the ancestral kinetoplastid (Skalický et al., 2017). There are two sets of microtubules, one a microtubule triplet and the other a microtubule quartet (distinct from the flagellum attachment zone MtQ) associated with the cytostome/cytopharynx complex. The cytostome/cytopharynx microtubule quartet is nucleated near the flagellar pocket and then extends out beyond the pocket, just under the cell membrane along the preoral ridge before dropping into the cytoplasm alongside the cytostome/cytopharynx. Conversely, the microtubule triplet is nucleated near the cytostome/cytopharynx entrance, and together, these two sets of microtubules form a V shape upon which the cytostome/ cytopharynx sits (Alcantara et al., 2014). In the latter stages of the cell cycle, during G 2 prior to flagellar pocket division, the cytostome/ cytopharynx complex and associated microtubules are disassembled, and then, the structure reassembles during late cytokinesis (Alcantara, L., Vidal, J.C., Souza, W. de, & Cunha-e-Silva, N.L., 2017).
Interestingly, it has also been shown that the MVT lysosome in dividing Leishmania cells also disassembles forming one or two sets of vesicles (Ilgoutz et al., 1999;Weise et al., 2000).
Here, we used cysteine peptidase A (CPA) and sperm flagellar 1 (SPEF1) as markers of the MVT lysosome and its associated microtubule, respectively, to characterize the cell cycle-related changes in these structures. We show that both the lysosome and its microtubule extend during G 1 /S phase of the cell but disassemble rapidly during G 2 and are essentially absent during cytokinesis before assembling again during the next G 1 . This cycle of assembly and disassembly is associated with a change in the endocytic capacity of the Leishmania cell.

| MVT lysosome disassembles prior to cell division
We have previously identified cysteine peptidase A (CPA) as a MVT lysosomal protein amenable to analysis by microscopy when tagged with a fluorescent protein (Halliday et al., 2019). We also determined that SPEF1, a protein originally identified as a proximal flagellum attachment zone MtQ-associated protein in T. brucei (Gheiratmand, Brasseur, Zhou, & He, 2013), localizes to an additional structure in L. mexicana which could plausibly be the lysosomal microtubule(s; Halliday et al., 2019). Previous work by other groups had indicated that there were cell cycle-related changes in the organization of the MVT lysosome (Ilgoutz et al., 1999;Weise et al., 2000). However, these studies had limited time resolution and only provided snapshots of the MVT lysosome and did not link these changes in MVT lysosome organization to the lysosomal microtubule(s) or lysosomal function. We wanted to examine these organizational changes, the interrelationships between them, and their functional consequences in detail using direct markers of these structures.
We generated cell lines expressing CPA and SPEF1 tagged with mNeonGreen (mNG) at their endogenous loci and examined their localization during the cell cycle (Figure 1a (Wheeler, Gluenz, & Gull, 2011). We measured cell body length and the length of the CPA::mNG labeled lysosome in 1F1K1N cells ( Figure 1b). There was a positive correlation between cell body length and CPA::mNG signal length, with CPA::mNG signal length increasing as cell body length increased. Moreover, the shorter cells at the start of the cell cycle tended to have the vesicular CPA::mNG signal, and this suggests that as the cell cycle progressed the lysosome switched from a vesicular to an extended tubular structure.
When we examined cells with two flagella, we again observed that cells either had a tubular (56%) or vesicular (44%) CPA::mNG signal ( Figure 1c). To determine the cell cycle position of the cells F I G U R E 1 Localization and morphological changes in CPA::mNG and mNG::SPEF1 throughout the cell cycle. Images of CPA::mNG (a) or mNG::SPEF1 (d) localization during the cell cycle in the Leishmania promastigote. Micrographs of major cell cycle stages, cells were ordered based on the number of kinetoplasts (K), nuclei (N), and flagella (F). Nuclear and kinetoplast DNA were labeled with Hoechst 33342. The scale bar represents 5 µm. Scatter plot of cell body length or new flagellum length (measured from cell tip to flagellum tip) against CPA::mNG (B) or mNG::SPEF1 (e) length in 1F or 2F stage, respectively. Each dot represents one cell, n = 303 for 1F and 101 for 2F CPA::mNG, n = 297 for 1F and 98 for 2F mNG::SPEF1. Bar charts of CPA::mNG (c) or mNG::SPEF1 (f) signal categories in 1F and 2F cells.
with two flagella, we measured the length of the new flagellum and correlated that with the length of the CPA::mNG signal ( Figure 1b).
In these cells, the CPA::mNG signal length remained relatively constant until the new flagellum reached ~4 µm long at which point the CPA::mNG signal began to shorten from the posterior end and disassembled into vesicles with no long tubule observed ( Figure 1a).
The disassembly correlated with the onset of cell division, and our images appeared similar to a dividing cell expressing GFP::DPMS (Ilgoutz et al., 1999). In the few cells in which the new flagellum was longer than ~7 µm, there was accumulation of CPA::mNG, causing an increase in the length of the CPA::mNG signal; however, this signal did not have a clear tubular structure. The increase in CPA::mNG is potentially an early step in the reassembly of the MVT lysosome.
In However, as the time course continued, FM4-64 did not progress to later endocytic compartments and remained associated with the flagellar pocket region as shown by its colocalization with SEC10 ( Figure A4). However, the colocalization of SEC10::mNG and FM4-64 was not complete, suggesting that the FM4-64 was internalized into the endocytic system surrounding the flagellar pocket but was not able to progress any further. Together, these data indicate that the rate of endocytosis in cells undergoing cytokinesis is greatly reduced.

| CON CLUS IONS
In many eukaryotes, the disassembly of organelles preceding cell di- During our search for lysosomal protein markers (Halliday et al., 2019), we noticed that the lysosomal protein p67 found in T. brucei and T. cruzi, which is related to the lysosome-associated membrane proteins found in the lysosome of many eukaryotes, is missing from the Leishmania genomes. In T. brucei, p67 has a function in maintaining the morphology of the lysosome and the loss of p67 in Leishmania might be associated with the unusual structure of the MVT lysosome (Peck et al., 2008).
The dynamic nature of the MVT lysosome has striking parallels with cytostome/cytopharynx of T. cruzi, which also undergoes a similar cell cycle regulation, disassembling before cytokinesis, and reassembling afterward (Alcantara et al., 2017). Moreover, the MVT lysosome and the cytostome/cytopharynx have a similar membrane tubule structure with associated microtubules which nucleate near the flagellar pocket. The cytostome/cytopharynx was likely an ancestral feature of kinetoplastids and given the similar cell cycle dynamics and overall architecture perhaps the MVT lysosome and lysosomal microtubule(s) are a reduced cytostome/cytopharynx-like feature (Skalický et al., 2017;Weise et al., 2000). However, there are significant differences in the orientation and path of the cytosome/ cytopharynx and lysosomal microtubule(s). We also found that when the MVT lysosome has disassembled, there was a dramatic reduction in the rate of endocytosis, which again was observed when the cytostome/cytopharynx disassembled in T. cruzi (Alcantara et al., 2017).
Here, we have provided insight into the cell cycle-dependent restructuring of the late endocytic system and the resulting effect on endocytic rate in Leishmania. The disassembly of the MVT lysosome is likely to be a critical step in Leishmania cell division and as such deciphering the regulation of this process within the context of the cell cycle will be an important step in understanding cell cycle coordination in these organisms. Our study highlights further commonalities and differences between the "TriTryps" and reinforces the added value that can be gained from comparative analyses of basic cell processes between the different kinetoplastids.

| Fluorescence microscopy and morphometric measurements
For live cell microscopy, cells were harvested by centrifugation at 800 g for 5 min and washed three times in PBS with Hoechst 33342 (1 μg/ml) in the first wash. The cells were resuspended in 30 μl PBS, and 1 μl was then placed on a microscope slide and immediately imaged using a Zeiss ImagerZ2 microscope with a 63 × NA 1.4 objective and Hamamatsu Flash 4 camera. Length measurements were made in ImageJ (Rueden et al., 2017).

| Pulse-chase endocytosis assay
Promastigotes (5 × 10 6 cells) were incubated in complete M199 medium on ice for 20 min before 40 μg/ml FM4-64 (Invitrogen) was added for 1 min. Cells were immediately harvested by centrifugation at 800 g and resuspended in prewarmed M199 with no dye at 28°C.
At each time point, cells were removed and washed with PBS before imaging. with a Gatan 3view2XP system. Serial images of the block face were recorded at an accelerating voltage of 1.5 kV, a spot size of 1 and an aperture size of 20 μm, and pressure of 0.0 Torr. Pixel size and the dwell time for each micrograph was 2 nm, 1 μs, and slice thickness was 75 nm. Images were recorded using Digital Micrograph. Threedimensional models were generated by tracing the SBFSEM images using IMOD (Kremer, Mastronarde, & McIntosh, 1996) and visualized using Blender. MVT lysosomes were distinguishable from glycosomes due to higher luminal electron density and greater length and distinguishable from mitochondrion branches due to lower membrane electron density than the double mitochondrial membrane.

ACK N OWLED G M ENTS
We are especially grateful to Keith Gull for his advice and guidance and laboratory space. We would like to thank Eva Gluenz (University

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All data are provided in full in the results section of this paper.
A PPE N D I X 1 F I G U R E A 1 Localization of CPA::mNG and mCh::SPEF1 in axenic amastigotes. Images of CPA::mNG (green) and mCh::SPEF1 (red) colocalization in axenic amastigotes. The scale bar represents 5 µm. White asterisk in the mCh::SPEF1 channel indicates the anterior end of the cell. mCh::SPEF1 localized to two structures a bright spot close to the flagellar pocket and a fainter curved line extending toward the posterior end of the cell. Two types of CPA::mNG localization pattern were observed, either a curved line extending from near the flagellar pocket to the posterior end of the cell or a series of variable sized points that followed a line through the cell. These large spots correlate with the megasomes previously observed by TEM (Waller & McConville, 2002). Both types of CPA::mNG localization pattern run alongside the mCh::SPEF1 signal, indicating a close association of these proteins in the amastigote form mCh::SPEF1 CPA::mNG mCh::SPEF1 CPA::mNG Merge F I G U R E A 2 Interpretation of cell structure from scanning block-face electron microscopy (relating to Figure 2). (a) Three-dimensional reconstructions of the major membrane-bound cytoplasmic structures, endoplasmic reticulum (orange), mitochondrion (blue), and MVT lysosome (purple), in an example 1K1N Leishmania promastigote from serial block-face scanning electron microscopy images. The MVT lysosome is a third extended membrane-bound network in addition to the endoplasmic reticulum and mitochondrion. (b) SBFSEM image illustrating identifiable structures in the cell. A combination of appearance (electron density) and 3D shape allow organelle identification. Acidocalcisomes and lipid droplets are near-spherical organelles with high and low electron density, respectively. Glycosomes are more elongated with intermediate electron density. The endoplasmic reticulum, nuclear envelope, mitochondrion, and MVT lysosome form extended tubes/networks. The endoplasmic reticulum and nuclear envelope have a narrow lumen and the two membranes are not well resolved, instead appearing as a single highly electron dense line. The nucleus is identifiable from the chromatin contents. The mitochondrion lumen is well resolved with cristae occasionally visible. The double membrane is not well resolved, again appearing as a single highly electron dense line. The MVT lysosome has a wider lumen than the endoplasmic reticulum and does not have lamellar regions. The MVT lysosome is bounded by a single membrane which appears less electron dense than the mitochondrion or endoplasmic reticulum membranes F I G U R E A 3 Localization of proteins identified in L. mexicana with identity to known regulators of lysosome function (relating to Table A1). Images of cell lines expressing identified proteins tagged at either the N-or C-terminus with mNG in Leishmania promastigotes. Nuclear and kinetoplast DNA were labeled with Hoechst 33342. The name of the human protein is on the right with the corresponding L. mexicana gene ID in white text in the mNG channel image. Beneath each set of images is the description of the protein localization. The proteins in bold are those cell lines which have a proportion of cells in which a lysosome localization was observed