4D live-cell imaging of microgametogenesis in the human malaria parasite Plasmodium falciparum

Formation of gametes in the malaria parasite occurs in the midgut of the mosquito and is critical to onward parasite transmission. Transformation of the male gametocyte into microgametes, called microgametogenesis, is an explosive cellular event and one of the fastest eukaryotic DNA replication events known. The transformation of one microgametocyte into eight flagellated microgametes requires reorganisation of the parasite cytoskeleton, replication of the 22.9 Mb genome, axoneme formation and host erythrocyte egress, all of which occur simultaneously in <20 minutes. Whilst high-resolution imaging has been a powerful tool for defining stages of microgametogenesis, it has largely been limited to fixed parasite samples, given the speed of the process and parasite photosensitivity. Here, we have developed a live-cell fluorescence imaging workflow that captures the explosive dynamics of microgametogenesis in full. Using the most virulent human malaria parasite, Plasmodium falciparum, our live-cell approach combines three-dimensional imaging through time (4D imaging) and covers early microgametocyte development through to microgamete release. Combining live-cell stains for DNA, tubulin and the host erythrocyte membrane, 4D imaging enables definition of the positioning of newly replicated and segregated DNA. It also shows the microtubular cytoskeleton, location of newly formed basal bodies and elongation of axonemes, as well as behaviour of the erythrocyte membrane, including its specific perforation prior to microgamete egress. 4D imaging was additionally undertaken in the presence of known transmission-blocking inhibitors and the untested proteasomal inhibitor bortezomib. Here, for the first time we find that bortezomib inhibition results in a clear block of DNA replication, full axoneme nucleation and elongation. These data not only define a framework for understanding microgametogenesis in general but also suggest that the process is critically dependent on proteasomal activity, helping to identify potentially novel targets for transmission-blocking antimalarial drug development.

shows the microtubular cytoskeleton, location of newly formed basal bodies and elongation 34 of axonemes, as well as behaviour of the erythrocyte membrane, including its specific 35 perforation prior to microgamete egress. 4D imaging was additionally undertaken in the 36 presence of known transmission-blocking inhibitors and the untested proteasomal inhibitor 37 bortezomib. Here, for the first time we find that bortezomib inhibition results in a clear block 38 of DNA replication, full axoneme nucleation and elongation. These data not only define a 39 framework for understanding microgametogenesis in general but also suggest that the 40 process is critically dependent on proteasomal activity, helping to identify potentially novel 41 targets for transmission-blocking antimalarial drug development. 42

INTRODUCTION 43
Malaria disease is caused by single-cell protozoan parasites from the genus Plasmodium. 44 Over its complex two-host lifecycle, the Plasmodium cell demonstrates remarkable cellular 45 plasticity as it transitions between multiple developmental stages. In the transition from 46 mammalian to mosquito host, the parasite faces an extreme population bottleneck in 47 numbers, which also presents a natural target for novel antimalarial treatments aimed at 48 blocking transmission. Transmission is triggered by the uptake of sexual stage gametocytes 49 during a mosquito feed that instantly activate, initiating a transformation in the mosquito 50 midgut that has become a trademark in the cellular biology of these protozoan parasites 1 . 51 Dormant male (micro) and female (macro) gametocytes form a sub-population of between 52 0.2-1% of the circulating asexual blood stage parasite reservoir in the mammalian host. The 53 signals that initiate commitment of asexual parasites to sexual differentiation are, however, 54 poorly understood 1 . Committed gametocytes mature over five distinct morphological stages 55 (referred to as stages I-V) and are believed to sequestrate in the host bone marrow and 56 spleen, before emerging into the bloodstream when they reach stage V maturity 1-3 . Following 57 ingestion by a feeding mosquito, stage V microgametocytes and macrogametocytes 58 transform rapidly to microgametes and macrogametes, respectively. The transformation from 59 gametocyte to gamete, a process termed gametogenesis, is activated by a decrease in 60 temperature to 20-25°C, rise in pH and the presence of the mosquito metabolite, xanthurenic 61 acid in the mosquito midgut 4 . 62 Plasmodium gametogenesis is distinctly different between male and female parasites. Both 63 entail a morphological change from falciform to rounded, in P. falciparum, and egress from 64 within the host erythrocyte by an 'inside-out' mechanism. This mechanism of egress involves 65 disintegration of the parasitophorous vacuole membrane (PVM) prior to that of the host 66 erythrocyte 5 . The female macrogametocyte rounds up 6 and egresses 7 within 10 minutes of 67 activation, emerging as a fertilisation competent macrogamete. Whilst this process is 68 incompletely understood, reverse genetic studies using different Plasmodium species have 69 the specificity of fluorescent imaging often requires antibody staining, limiting imaging to 97 fixed samples. This is also true for electron microscopy, despite its proven utility in shaping 98 our current understanding of the fine cellular biology of microgametogenesis 14,15,23 . As a 99 result, the dynamic nature of events encompassing microgametogenesis are still very poorly 100 understood. Better temporal characterisation using live samples, coupled with the specificity 101 of fluorescently tagged structures would allow a marked improvement in our understanding 102 of the process of microgametogenesis and provide a platform from which strategies to block 103 it might then be translatable. 104 Low and high-resolution microscopy of Plasmodium has been extensively used to 105 understand the cell biology of parasite development and aid drug-intervention studies. 106 Ultrastructure expansion microscopy was recently shown to advance traditional fluorescence 107 microscopy approaches to fixed parasite imaging, allowing close observation of asexual 108 blood stage, microgametocyte and ookinete cytoskeletal development 22 . A recent study 109 reported the application of semi-supervised machine learning to define asexual parasite 110 development in a high-throughput imaging format, using fixed parasites 24 . The study proved 111 to be a powerful tool in detecting parasites with morphological perturbations when treated 112 with known antimalarials 24 . Another recent high throughput screen reported the phenotypes 113 of transmission blocking antimalarial hits with unknown cellular targets 25 . The study utilised 114 immunofluorescence labelling of fixed parasites undergoing microgametogenesis to 115 manually define cellular phenotypes. The same screen utilised low-resolution, live brightfield 116 imaging of exflagellation in a high-throughput assay 21 to identify the microgametogenesis-117 blocking hits 25 . Live-cell fluorescence microscopy has also been explored, with a recent 118 study utilising lattice light-sheet microscopy to acquire 3D live time-lapse data of asexual P. 119 falciparum invasion 26 . Other studies have reported the use of live-cell fluorescence imaging 120 of microgametogenesis to define the phenotypes of transgenic P. berghei 27,28 cell lines. 121 Given the error prone nature of microgametogenesis 16 , however, it is important to define true 122 perturbations to microgametogenesis over natural variation and, critically, to do so in real 123

time. 124
Here, we describe a protocol that enables the labelling of microtubules, DNA and host 125 erythrocyte membrane for P. falciparum microgametocytes and their imaging by live-cell 3D 126 fluorescence microscopy (4D imaging), capturing the entire process of microgametogenesis. 127 To develop a workflow that is translatable to other research labs, we have used a 128 combination of widefield microscopy, an open-source analysis software for deconvolution 129 and commercially available reagents throughout this study. Using this approach, we define in 130 detail the dynamic morphological transformations that occur during microgametogenesis, 131 from activation through to exflagellation. Furthermore, we demonstrate the applicability of 132 our protocol to phenotypic characterisation of inhibitors of microgametogenesis, in particular 133 the role of the proteasome, demonstrating the power of this approach for future 134 transmission-blocking drug discovery 24 . 135

Development of a live microgametogenesis 4D imaging approach 137
To date, visualisation of the complex cytoskeletal rearrangement, host erythrocyte egress 138 and DNA replication events during P. falciparum microgametogenesis ( Figure 1A) has 139 mostly been limited to fixed imaging protocols. We set out to devise a live cell imaging 140 workflow ( Figure 1B DyeCycle™ Violet is a cell permeable dye which binds to double-stranded DNA to emit a 149 fluorescent signal proportional to DNA mass and has been previously used to measure 150 microgametocyte genome replication during microgametogenesis 31,32 using flow cytometry. 151 Stage V gametocytes from the P. falciparum NF54 strain, were cultured as previously 152 described 33 , stained and strictly maintained at 37°C to prevent premature activation of 153 gametogenesis. Gametogenesis was initiated by mimicking conditions of the mosquito 154 midgut using "ookinete media" (see Materials and Methods), a xanthurenic-acid-containing 155 media maintained at pH 7.4 and used at room temperature (RT). Labelled gametocytes were 156 directly added to ookinete media-containing imaging slides and prepositioned on the 157 microscope for the immediate acquisition of time-lapse data ( Figure 1B). 158 To visualise the initial developmental stages of microgametogenesis, microgametocytes 159 were identified by SiR-tubulin-stained mitotic spindles which signified a successful round of 160 DNA replication ( Figure 1A). Due to the rapid turnaround between activation and DNA 161 replication, most time-lapses presented here were acquired from 1-2 minutes-post activation, 162 following a round of replication. In optimisation of the imaging workflow, we found the 163 alternation between fluorescence and brightfield acquisition to significantly maximise the 164 viability of microgametocytes. Following identification of an activated microgametocyte, 3-165 colour fluorescent time-lapses were immediately acquired before switching to brightfield 166 microscopy. A maximum of 10 frames were acquired in fluorescence to minimise the 167 phototoxic effects and brightfield microscopy was subsequently used to monitor parasite 168 development. Upon observation of further parasite differentiation in brightfield, for example 169 by rounding up or preparing for egress, image acquisition was switched back to fluorescence 170 for further acquisition of time-lapses to capture the full early developmental stages of 171

microgametogenesis. 172
Despite best efforts to reduce LED intensity and frame rates, phototoxicity nearly always 173 prevented the complete visualisation of microgametogenesis from start to finish. To 174 circumvent this issue, activated microgametocytes were imaged at different stages to ensure 175 full capture of later developmental stages, specifically the emergence of microgametes 176 during exflagellation ( Figure 1A). In this late-stage instance, viable microgametocytes were 177 identified based on SiR-tubulin-stained axonemes, coiled around the parasite cell body. 178 Whilst the earlier stages of microgametogenesis were acquired in 4D, through Z and T, 179 exflagellation could only be captured as single Z-slice time-lapses given the dynamic nature 180 of emerging microgametes ( Figure 1B). Time-lapse data of the early and later stages of 181 microgametogenesis were subsequently combined and could be analysed together, enabling 182 us to dissect microgametogenesis in its entirety, from initial endomitotic division through to 183 microgamete emergence, for the first time. 184 185

Insights into cytoskeletal rearrangements during microgametogenesis 186
The formation of mitotic spindles, basal bodies and axonemes occurs with rapid succession 187 during the early stages of microgametogenesis 15,16 . Using our 4D imaging platform we 188 sought to define these stages in real time.   The host erythrocyte perforates and forms a pore for microgametocyte egress 240 A key stage in microgametogenesis is parasite egress from the host erythrocyte ( Figure 2C, 241 E and Supplementary Video 3). 4D imaging revealed that the erythrocyte membrane 242 perforated in preparation for egress (Figure 2D and 5A). Activated microgametocytes 243 aligned at the periphery of the parasite membrane, eventually ejecting from a frequently 244 singular pore that formed in the host erythrocyte ( Figure 2E and Supplementary Video 3). 245 Notably, we observed microgametocytes eject from a spindle pole of the parasite, out of the 246 newly formed host erythrocyte pore ( Figure 2E, Figures S1B-D, Figure 5A to earlier stages of microgametogenesis (Figure 2B-C and 3), signifying successful 267

replication. 268
Due to the dynamic nature of emerging microgametes that were motile through Z-269 acquisition, 2D rather than 3D time-lapse data was acquired during exflagellation. Although 270 3D frames of exflagellation were not obtained, it is possible to obtain 3D plots of SiR-tubulin 271 intensity staining (Figure 3D). This allowed the identification of dense regions of SiR-tubulin 272 staining resulting from axoneme overlap, which would otherwise not be deducible without 4D 273 imaging ( Figure 3D). Alternatively, as parasite motion halts during loss of viability, 3D data 274 can be obtained to closely observe the positioning of emerged microgametes ( Figure 3E

Drug inhibition of microgametogenesis 279
The process of microgametogenesis is tightly synchronised by a series of cell cycle 280 regulators and is consequently sensitive to and the target of known and developmental drug 281 treatments 25 . We sought to apply our 4D imaging approach as a drug discovery tool that 282 could help elucidate the cellular phenotypes of compounds with known and unknown activity 283 against microgametogenesis regulators towards defining their mode or process of action. Using 4D imaging, we could resolve distinct cellular phenotypes for each drug and its target 292 ( Figure 4A-B, D-H and Supplementary Videos 18-24). 293 CDPK4 inhibition by 1294 31,34 prevented morphological transformation from falciform to 294 round ( Figure 4A and G), DNA replication ( Figure 4F) and microtubule polymerisation 295 ( Figure 4D-E) during microgametogenesis (Supplementary Videos 18-22). On detailed 296 inspection, 1294-treated parasites, failed to reach the maximum level of cell circularity 297 ( Figure 4G) and SiR-tubulin intensity (Figure 4A, D, E and G), indicating a role of CDPK4 in 298 microgametocyte rounding as well as cytoskeletal rearrangement during 299 microgametogenesis. Of note, many 1294-treated microgametocytes were observed to have 300 egressed from the onset of activation, a probable stress-response of CDPK4 inhibition 301 ( Figure 4A). Of the population of 1294-treated microgametocytes, 50% were able to fully 302 egress compared to the 53% of untreated microgametocytes which egressed from the host 303 erythrocyte (Figure 4H). An incomplete egress phenotype in which falciform parasites 304 partially emerged from the host erythrocyte was also observed, with 15% of 1294-treated 305  Figure 4F) and microtubule polymerisation (Figure 4D and E). 316

ML10-treated gametocytes retained a falciform morphology and a level of SiR-tubulin 317
staining on par with that seen at the onset of gametogenesis activation (Figure 4D-E and G  318 and Supplementary Videos 23-24). SiR-tubulin intensity was significantly lower than 319 untreated microgametocytes at 10 minutes post-activation ( Figure 4E). The host erythrocyte 320 egress phenotype was mixed, as 50% of ML10-treated cells emerged fully, 50% failed 321 (Figure 4Bii) and 8% partially emerged (Figure 4Bi) from the host erythrocyte (Figure 4A, 322 H and Supplementary Videos 23-24). We can deduct from these images that PKG plays a 323 significant role in regulating MTOC transformation, axoneme nucleation and elongation, DNA 324 replication and rounding up exhibited in microgametogenesis, as summarised in Figure 5C. able to nucleate axonemes from one end of the parasite, but no more than 3 axonemes were 347 formed and growth was truncated (Figure 4Cii-iii and Supplementary Videos 25-28). This 348 perturbation to axoneme nucleation and elongation resulted in a significant decrease in SiR-349 tubulin staining intensity (Figure 4D, E and G), although less significant than the decrease 350 observed with ML10 and 1294 treatment which blocked all microtubule polymerisation. 351 Proteasome-inhibited microgametocytes were, however, able to transform from falciform to 352 round ( Figure 4G and Supplementary Videos 25-28). 353 Bortezomib treatment also significantly reduced Vybrant™ DyeCycle™ Violet staining 354 intensity, signifying a probable indirect inhibitory effect on DNA replication (Figure 4F). 355 Additionally, we observed incomplete transformation of the MTOC which resulted in 356 truncated formation of few axonemes from one pole of developing microgametocytes. 357 Combined these data represent the first time that the cellular role of the proteasome during 358 microgametogenesis has been explored. Our findings, summarised in Figure 5C, add further 359 weight to bortezomib's use as a desirable antimalarial drug candidate that is able to inhibit 360 the sexual stages of Plasmodium in additional to asexual replication, representing the 361 potential to treat symptoms and block transmission with a single compound. fluorescence which may otherwise be missed with fixed or brightfield imaging. The data 416 depicted here promises to unveil novel insights into P. falciparum microgametogenesis for 417 cell biology and drug study, but the protocol is not limited to this. Cultivation of in vitro 418 Plasmodium cultures, at any stage, permits the live microscopy of the breadth of malaria 419 parasite development, from macrogametogenesis and asexual blood stage development to 420 liver stages. Additional stains for intracellular organelles, parasite membranes and sex-421 specific proteins, with both wild-type and transgenic lines, will now be a priority for exploring 422 so that we can shed further light on this ancient but deadly single-celled parasite. 423