Mutant/mutation
The mutant lacks expression of DHC3

Protein (function)
There are 7 genes encoding putative dynein heavy chain (DHC) proteins of Plasmodium parasites. In the rodent malaria parasite P. yoelii, they are PY17X_0418900 (dhc1), PY17X_0618400 (dhc2), PY17X_0215400 (dhc3), PY17X_0508400 (dhc4), PY17X_0927400 (dhc5), PY17X_0603800 (dhc6), and PY17X_1333900 (dhc7).
Each dynein contains at least one dynein heavy chain (DHC) and other subunits including the intermediate chain (IC), light intermediate chain (LIC), and light chain (LC).

The invasive “zoite” stages of Plasmodium, including the ookinete, sporozoite, and merozoite, are morphologically polarized and possess a unique cortical pellicle underneath the parasite plasma membrane. From outside to inside, the pellicle consists of a double membrane organelle inner membrane complex (IMC) and a cytoskeleton layer of apically radiating subpellicular microtubules (SPMTs), both of which associate with each other and span along the periphery of the zoite parasites. Besides SPMTs, the invasive zoites of apicomplexan parasites possess a highly specialized structure called the apical polar ring (APR) at the cell apical cortex. APR is recognized as an electron-lucent region beneath the apical IMC. it is believed that APR functions as a microtubule-organizing center (MTOC) for nucleating SPMTs at the Plasmodium zoites. In Plasmodium, the SPMT cytoskeleton functions as a scaffold supporting parasite morphogenesis, maintaining the polarized cell shapes and providing parasite rigidity during gliding and invasion. In the process of ookinete growth, the parasite undergoes massive expansion of the plasma and cortex membrane. In addition, the ookinete acquires a complete set of apical organelles and structures via de novo assembly. IMC is assembled at the apical site of the initial protrusion and extends along the expanding plasma membrane to the basal end. After biogenesis,APR nucleates the assembly of apical SPMTs underling the IMC. Meanwhile, the apical tubulin ring (ATR), another compacted structure of MTs, emerges at the apex of ookinetes.While the IMC, SPMT, APR, ATR, and microneme are essential for either development, gliding, or midgut invasion of ookinetes, the mechanisms for de novo assembly of these organelles and structures at the apical distal area are largely unknown. We hypothesize that besides playing a cytoskeleton role, the apically radiating SPMTs may function as the tracks for the apical transport of cargoes containing the contents required for the assembly of apical organelles and structures in the ookinetes. In this scenario, the cytoplasmic dynein could be the primary motor moving towards the minus end of SPMTs for cargo transport to the apical distal area of ookinetes. However, the SPMT-based dynein transport machinery has not been identified in Plasmodium. Cytoplasmic dynein is a large multi-subunit protein complex, and the core is a homodimer of two heavy chain subunits (DHCs) interacting with intermediate, light intermediate, and light chain subunits

Phenotype
To elucidate the function of DHC3 a 1.0 kb genomic sequence was deleted at the coding region (15.3 kb) of dhc3. The deletion caused a frameshift for the remaining coding sequence. deleted a 1.0 kb genomic sequence at the coding region (15.3 kb) of dhc3 in the 17XNL (wild type or WT) using CRISPR-Cas9. The deletion caused a frameshift for the remaining coding sequence.
Normal development/growth of asexual blood stages of Δdhc3 and and gametocyte formation in mice. The Δdhc3 showed normal gamete formation and fertilization in vitro. and developed from diploid to tetraploid during ookinete development. The in vitro assay for zygote to ookinete differentiation revealed that Δdhc3 had a dramatic decrease in ookinete formation (59% in 17XNL, 14% in Δdhc3). Time-course analysis revealed that DHC3 deficiency caused developmental arrestment mainly at stages I and II, and a small proportion of parasites developed into mature looking ookinetes. The defective morphology of the Δdhc3 ookinetes was also observed under scanning electron microscopy (SEM). Compared to the 17XNL ookinetes with characteristic crescent shapes, the mature-looking ookinetes of Δdhc3 lost cell bending.We further assessed the gliding activity of ookinetes in vitro and found that the mature-looking ookinetes of Δdhc3 displayed a significantly reduced gliding speed (17XNL: 8.4 ± 1.7 μm/min, n= 24; Δdhc3: 0.3 ± 0.2 μm/min, n = 20)
No oocyst formation. No salivary gland sporozoites.

GAP45 staining showed that DHC3 deletion had less effect on the IMC assembly in the Δdhc3-defective ookinetes . However, the in vitro assay for zygote to ookinete differentiation etc etc

Analysis of a mutant expressing a C-ternminal 6xHA-tagged version of DHC3 (RMgm-5615) showed the following:
No expression in asexual blood stages. Expression in female gametocytes, ookinetes and sporozoites. No expression in male gametocytes and oocysts. DHC3 was evenly distributed along the periphery of ookinetes.

Additional information
The seven dhc-genes were tagged with a sextuple HA epitope (6HA) to analyse the
expression and localization of these proteins.
See the mutants RMgm-5613 (PY17X_0418900-6HA; dhc1), RMgm-5614 (PY17X_0618400-6HA; dhc2), RMgm-5615 (PY17X_0215400-6HA; dhc3, RMgm-5616 (PY17X_0508400-6HA; dhc4), RMgm-5617 (PY17X_0927400-6HA; dhc5), RMgm-5618 (PY17X_0603800-6HA; dhc6), RMgm-5619 PY17X_1333900-6HA; dhc7). These 6HA-tagged parasite lines showed normal asexual blood stage proliferation and gametocyte differentiation in mice, suggesting that the addition of 6HA did not affect parasite viability. Immunofluorescence assay (IFA) showed that all 7 DHCs displayed no detectable expression in the asexual blood stages. Only DHC3 was expressed in female gametocytes, ookinetes, and sporozoites. The other 6 DHCs were specifically expressed in male gametocytes.

DHC3 was evenly distributed along the periphery of ookinetes, suggesting the existence of SPMT-based dynein in the ookinetes.
The localization of DHC3 was analysed relative to proteins known to be expressed within specific localizations in ookinetes. Parasite clones were generated with additional proteins tagged with quadruple Myc epitope (4Myc) or triple V5 epitope (3V5) from the dhc3::6HA parasite. These proteins included P28 (plasma membrane), GAP45 (IMC), MyosinB and SAS6L (apical tubulin ring, ATR), APR2 (apical polar ring, APR), GCβ (ookinete extrados site, OES), and CTRP and chitinase (microneme). Among these proteins, P28 and GAP45 showed overlapping signals with DHC3, further supporting the peripheral localization of DHC3 in the ookinetes.
To visualize DHC3 localization in living ookinetes, a parasite line was generated, dhc3::mScarlet, with DHC3 C-terminally tagged with a red fluorescence protein mScarlet (see mutant RMgm-5621). The mScarlet-tagged DHC3 was also distributed along the periphery of ookinetes.These results suggested the existence of the SPMT-based cytoplasmic dynein in the ookinetes and DHC3 is likely a subunit of the dynein complex.

The microtubule-binding domain (MTBD), a globular fragment of 80-130 amino acids in the stalk tip, is responsible for the MT binding of DHC4. To further validate the association of DHC3 with SPMTs, we deleted the MTBD and investigated the effect on the SPMT. localization of DHC3. In the P. yoelii, the MTBD (3559-3648 amino acid) is located between the AAA4 and AAA5 of the pseudo-hexameric ring composed of 6 ATPase modules.
We used CRISPR-Cas9 to delete the genomic sequences encoding MTBD of DHC3 in the dhc3::6HA parasite and obtained a mutant line designated as ΔMTBD (RMgm-5622). Removal of MTBD had little effect on the protein level of DHC3 in the gametocytes, however, ΔMTBD showed reduced  formation of mature ookinetes compared to the parental line. The truncated DHC3 lost the peripheral localization in the ΔMTBD ookinetes.

Component subunits of the SPMT-dynein complex in ookinetes
Cytoplasmic dynein comprises six subunits, including one HC, one IC, one LIC, and three LCs (Robl, LC8, and Tctex). We sought to identify all the subunits of SPMT cytoplasmic dynein in the ookinetes. In the P. yoelii genome, one putative dynein IC gene (PY17X_0505600), one putative dynein LIC gene (PY17X_0417700), and 12 putative dynein LC genes are encoded.
These 14 proteins (PY17X_1024300 Light chain (LC),  PY17X_1208500 Light chain (LC),  PY17X_0708400 Light chain (LC),  PY17X_1135100 Light chain (LC), PY17X_0302800 Light chain (LC),  PY17X_1241500 Light chain (LC),  PY17X_0935600 Light chain (LC),  PY17X_0215300 Light chain (LC), PY17X_0831600 Light chain (Tctex), PY17X_1431600 Light chain (LC8),  PY17X_0505400 Light chain (Robl),  PY17X_1040900 Light chain (LC),  PY17X_0505600 Intermediate chain (IC),  PY17X_0417700 Light Intermediate chain (LIC)), conserved in human and rodent Plasmodium species, have not been investigated for expression and localization. We tagged each of these candidate proteins with a 6HA at the C-terminus in the 17XNL using CRISPR-Cas9 and analyzed their expression and localization in ookinetes. IFA of the tagged parasite clones showed that five proteins (LIC, PY17X_0417700 RMgm-5623; IC, PY17X_0505600 RMgm-5624; three LCs, PY17X_0505400 RMgm-5625, PY17X_1431600 RMgm-5626, and PY17X_0831600 RMgm-5627) were localized at the periphery of ookinetes. In addition, these five proteins displayed similar localization dynamics during ookinete morphogenesis as DHC3 and were not detected in the asexual blood stages. To confirm the co-localization of these candidate subunits with DHC3, we engineered parasite clones with each of these five proteins tagged with a 6HA from the 4Myc::dhc3 parasite and obtained five double-tagged parasite lines. Two-colored IFA showed these five proteins were co-localized with DHC3 in the periphery of ookinetes. To visualize the association of the protein with SPMTs in more detail, the ookinetes were stained with antibodies against HA and α/β-Tubulin and imaged by U-ExM. These subunits (LIC, IC, Robl, LC8, and Tctex) showed complete co-localization with SPMTs in the ookinetes. It is noted that these five subunits showed no signal at the ATR, consistent with the results of DHC3. These pieces of evidence indicate that the SPMT-based cytoplasmic dynein complex is composed of DHC3, LIC, IC, and three LCs (Robl, LC8, and Tctex).

Impaired apical polar ring (APR) in the later-arrested ookinetes of Δdhc3.
Besides SPMTs, the invasive zoites of apicomplexan parasites possess a highly specialized structure called the apical polar ring (APR) at the cell apical cortex. APR is recognized as an electron-lucent region beneath the apical IMC, and resembles a cap-like structure in the transmission electron micrograph of the ookinetes. Since the minus-ends of all SPMTs emanate from APR, it is believed that APR functions as a microtubule-organizing center (MTOC) for nucleating SPMTs at the Plasmodium zoite.
In TEM micrographs of 17XNL ookinetes, APR is adjoined with apical IMC.However, APR appeared posteriorly at a distance from the apical IMC in the Δdhc3 ookinetes, suggesting APR detachment fromthe apical IMC. In addition, the detached APR appeared structurally defective.  ExM analysis of ookinetes after staining with NHS-ester dye also revealed the impaired APR in the Δdhc3 ookinetes. To visualize the defects of APR in more detail, we deleted the dhc3 gene in two parasite lines apr2::4Myc and ara1::4Myc13, in which the APR proteins APR2 (PY17X_1339500) and ARA1 (PY17X_1412750) were tagged with a 4Myc. In both mutant parasites apr2::4Myc;Δdhc3 and ara1::4Myc;Δdhc3, most of the defected ookinetes retained the apical localization of APR2 and ARA1. However, the IFA signals of APR2 and ARA1 were decreased in the ookinetes of the mutant compared to the parental line. U-ExM further confirmed the impaired APR in the later-arrested ookinetes
of apr2::4Myc;Δdhc3.

Small GTPases Rab11A and Rab11B co-localize and interact with DHC3
Small GTPases of the Rab family participate in cargo transport via vesicle trafficking in the eukaryotes. A recent study demonstrated the essential role of Rab11A in the P. berghei ookinete development. Knockdown of rab11a via promoter swap strategy inhibited ookinete development, and most of the ookinetes failed to elongate after protrusion. The Rab11A-deficient defects resembled the phenotype of the DHC3 null parasite, prompting us to explore the potential association between Rab proteins and DHC3 in ookinete development. Plasmodium encodes eleven Rab proteins. Among them, Rab1A (PY17X_1114600), (Rab7 PY17X_0421000), Rab11A (PY17X_1420600), Rab11B (PY17X_1359500), and Rab18 (PY17X_1226500) displayed relatively high levels of transcripts in gametocytes from previous transcriptome profiles. We investigated the expression and localization of these five Rab proteins in the ookinetes. Since the C-terminal modification of Rab would inactivate the protein, we failed to tag the endogenous protein at the C-terminus after several attempts. Alternatively,the expression cassettes of rab genes were integrated into the p230p locus using CRISPR-Cas9 in the dhc3::6HA parasite for transgenic overexpression. Each gene was tagged with a 4Myc at the N-terminus and driven by the hsp70 5′-UTR and the dhfr 3′-UTR. Five double-tagged parasite lines (dhc3::6HA;4Myc::rab1a, dhc3::6HA;4Myc::rab7, dhc3::6HA;4Myc::rab11a, dhc3::6HA;4Myc::rab11b, and dhc3::6HA;4Myc::rab18) were obtained. IFA showed that Rab11A and Rab11B were primarily distributed at the periphery and colocalized with DHC3 in ookinetes of the dhc3::6HA;4Myc::rab11a (DTS1) and dhc3::6HA;4Myc::rab11b (DTS2) lines, respectively. Rab1A, Rab7, and Rab18 appeared to be cytoplasmic. Rab11A localization is in agreement with that of Rab11A detected by antiserumin the P. berghei ookinete45. Time-course analysis of ookinete development showed that Rab11A and Rab11B displayed similar localization patterns as DHC3. Peripheral co-localization with DHC3 suggested that Rab11A and Rab11B associate with dynein along the SPMTs. These results demonstrated that Rab11A and Rab11B are associated with the DHC3-residing dynein which tracks along the SPMTs.
Localization of Rab11A and Rab11B to SPMTs requires DHC3 but not vice versa To investigate whether SPMT localization of Rab11A and Rab11B is dependent on DHC3, we deleted the dhc3 gene in the dhc3::6HA;4-Myc::rab11a (DTS1) and dhc3::6HA;4Myc::rab11b (DTS2) parasites, generating two mutant lines DTS1;Δdhc3 and DTS2;Δdhc3. DHC3depletion did not affect the protein amount of Rab11A and Rab11B in either gametocytes or early ookinetes, ruling out an effect of DHC3 on protein synthesis of Rab11A and Rab11B. In the paper evidence is presented that 'disturbing Rab11A (PY17X_1420600) or Rab11B (PY17X_1359500)) phenocopies DHC3 deficiency in ookinete morphogenesi'.

From the paper:
The cortical cytoskeleton of subpellicular microtubules (SPMTs) supports the Plasmodium ookinete morphogenesis during mosquito transmission of malaria. SPMTs are hypothesized to function as the cytoskeletal tracks in motor-driven cargo transport for apical organelle and structure assembly in ookinetes. We identify the core subunit DHC3 and other subunits of dynein showing co-localizing with SPMTs in the ookinete. In-depth phenotypical and functional analyses demonstrate that SPMT-based dynein plays an essential role in ookinete morphogenesis, shape, and gliding motility. DHC3 disruption impairs ookinete development, shape, and gliding, leading to failure in mosquito infection of Plasmodium. The DHC3-deficient ookinetes display defective formation or localization of apical organelles and structures. Rab11A and Rab11B interact with DHC3 at SPMTs in a DHC3-dependent manner, likely functioning as the receptors for the cargoes driven by SPMT-dynein. Disturbing Rab11A or Rab11B phenocopies DHC3 deficiency in ookinete morphogenesis. This study confirms the existence of the SPMT-based cytoplasmic dynein motor in the ookinetes and reveals its importance in intracellular cargo transport in ookinete morphogenesis.

Other mutants