Chhitar Mal Gupta

The actin cytoskeleton plays a crucial role in fundamental eukaryotic processes such as morphogenesis, motility, endocytosis, intracellular trafficking, and cell division. However, our understanding of actin and its associated proteins in trypanosomatid parasites like Leishmania remains limited. Over the past two decades, considerable progress has been made in elucidating the structure and functions of Leishmania actin and its core regulators. Notably, these findings are primarily derived from studies of the insect-stage promastigote form, while the roles of the actin machinery during the disease-causing amastigote stage within mammalian hosts remain largely unexplored. This review consolidates the current knowledge of actin and its interactors in Leishmania promastigotes, highlighting their potential roles in parasite development and stage-specific differentiation. Additionally, it explores the potential of targeting the cytoskeletal system as a strategy for novel therapeutic interventions against Leishmaniasis. The review concludes by identifying critical knowledge gaps and proposing future research directions to better understand actin-driven pathogenesis in this important human parasite.

Profilins are actin binding proteins that play a central role in regulation of actin remodeling in all eukaryotic organisms. Leishmania, a family of protozoan parasites that cause leishmaniasis, express a single homolog of profilin, which differs from other eukaryotic profilins in that it contains an extra stretch of 20 amino acids (actin binding domain) through which it directly binds actin. We therefore considered it of interest to analyze the role of direct binding of profilin with actin in interactions and functions of profilin in L. donovani promastigotes. For this, we deleted the actin binding domain in L. donovani profilin (LdPfn), and then carried out comparative analysis of LdPfn and truncated LdPfn (δLdPfn) interactomes by affinity pull-down and mass spectrometry. To assess the effect of deletion of the actin binding domain on the LdPfn functions, we expressed GFP conjugates of LdPfn and δLdPfn in the wild type Leishmania cells and then carried out comparative analysis of their growth, cell division cycle and intracellular vesicle trafficking activity. Results revealed that expression of GFP-δLdPfn in the wild type cells adversely affected their cell growth, intracellular vesicle trafficking and G1-to-S and S-to-G2/M phase transitions during their cell division cycle. Also, there was a complete loss of LdPfn binding to several cellular proteins including actin and mitochondrial outer membrane protein, porin (LdPorin) after deleting its actin binding domain. Further, to assess the effect of lack of LdPorin binding to δLdPfn on the mitochondrial functions, we measured the cellular ATP levels in wild-type and transgenic promastigotes. The ATP levels were significantly increased by expressing GFP-δLdPfn in wild-type cells. These results taken together strongly indicate that LdPfn-driven actin remodeling besides playing a pivotal role in regulation of intracellular vesicle trafficking and cell division cycle, it also plays an important role in regulation of Leishmania mitochondrial activity.

Profilin is a multi-ligand binding protein, which is a key regulator of actin dynamics and involved in regulating several cellular functions. It is present in all eukaryotes, including trypanosomatids such as Leishmania. However, not much is known about its functions in these organisms. Our earlier studies have shown that Leishmania parasites express a single homologue of profilin (LdPfn) that binds actin, phosphoinositides and poly- L- proline motives, and depletion of its intracellular pool to 50%of normal levels affects the cell growth and intracellular trafficking. Here, we show, employing affinity pull-down and mass spectroscopy, that LdPfn interacted with a large number of proteins, including those involved in mRNA processing and protein translation initiation, such as eIF4A1. Further, we reveal, using mRNA Seq analysis, that depletion of LdPfn in Leishmania cells (LdPfn^+/-) resulted in significantly reduced expression of genes which encode proteins involved in cell cycle regulation, mRNA translation initiation, nucleosides and amino acids transport. In addition, we show that in LdPfn^+/- cells, cellular levels of eIF4A1 protein were significantly decreased, and during their cell division cycle, G1-to-S phase progression was delayed and orientation of mitotic spindle altered. These changes were, however, reversed to normal by episomal expression of GFP-LdPfn in LdPfn^+/- cells. Taken together, our results indicate that profilin is involved in regulation of G1-to-S phase progression and mitotic spindle orientation in Leishmania cell cycle, perhaps through its interaction with elF4A1 protein.

Leishmania genome encodes for two isoforms of myosin, but only Myosin XXI (Myo21), which is a novel form of myosin in that it contains two ubiquitin associated-like (UBA) domains towards the end of its tail structure, is expressed in both the promastigote and amastigote forms of this protozoan. Earlier studies have shown that in Leishmania promastigotes Myo21 besides localizing throughout the cell body and flagellum, it is prominently localized to the base of the flagellum. It has further been shown that this protein in the promastigotes plays an important role in regulating the cell morphology, motility, flagellum dynamics, growth and intracellular trafficking, As Myo21 depletion has been shown to result in reduced cell growth in culture, we considered it of interest to investigate whether the observed effect of Myo21 on the cell growth is mediated through its possible role in Leishmania cell division cycle. For this, we prepared heterozygous Myo21 mutants of Leishmania promastigotes (Myo21 ^+/− cells) and then analyzed their morphology, growth and cell division cycle, using wild type Leishmania promastigotes (Myo21 ^+/+ cells) as control. The cell division cycle was analyzed by employing flow cytometry and immunofluorescence microscopy. Flow cytometric analysis revealed that the G2/M to G1 phase transition in Myo21 ^+/− cell is significantly delayed, as compared to Myo21 ^+/+ cells. Immunofluorescence confocal microscopic analysis indicated that Myo21 ^+/− cells encountered a significant delay in initiation of cytokinesis, which was mainly due to delay in the flagellar pocket division. Further analysis revealed that actin-based Myo21 motor is essentially required in the initiation phase of Leishmania cytokinesis.

Actin is the major protein constituent of the cytoskeleton that performs wide range of cellular functions. It exists in monomeric and filamentous forms, dynamics of which is regulated by a large repertoire of actin binding proteins. However, not much was known about existence of these proteins in trypanosomatids, till the genome sequence data of three important organisms of this class, viz. Trypanosoma brucei, Trypanosoma cruzi and Leishmania major, became available. Here, we have reviewed most of the findings reported to date on the intracellular distribution, structure and functions of these proteins and based on them, we have hypothesized some of their functions. The major findings are as follows: (1) All the three organisms encode at least a set of ten actin binding proteins (profilin, twinfilin, ADF/cofilin, CAP/srv2, CAPz, coronin, two myosins, two formins) and one isoform of actin, except that T. cruzi encodes for three formins and several myosins along with four actins. (2) Actin 1 and a few actin binding proteins (ADF/cofilin, profilin, twinfilin, coronin and myosin13 in L. donovani; ADF/cofilin, profilin and myosin1 in T. brucei; profilin and myosin-F in T.cruzi) have been identified and characterized. (3) In all the three organisms, actin cytoskeleton has been shown to regulate endocytosis and intracellular trafficking. (4) Leishmania actin1 has been the most characterized protein among trypanosomatid actins. (5) This protein is localized to the cytoplasm as well as in the flagellum, nucleus and kinetoplast, and in vitro, it binds to DNA and displays scDNA relaxing and kDNA nicking activities. (6) The pure protein prefers to form bundles instead of thin filaments, and does not bind DNase1 or phalloidin. (7) Myosin13, myosin1 and myosin-F regulate endocytosis and intracellular trafficking, respectively, in Leishmania, T. brucei and T. cruzi. (8) Actin-dependent myosin13 motor is involved in dynamics and assembly of Leishmania flagellum. (9) Leishmania twinfilin localizes mostly to the nucleolus and coordinates karyokinesis by effecting splindle elongation and DNA synthesis. (10) Leishmania coronin binds and promotes actin filament formation and exists in tetrameric form rather than trimeric form, like other coronins. (11) Trypanosomatid profilins are essential for survival of all the three parasites.

Myosin XXI (Myo21) is a novel class of myosin present in all kinetoplastid parasites, such as Trypanosoma and Leishmania. This protein in Leishmania promastigotes is predominantly localized to the proximal region of the flagellum, and is involved in the flagellum assembly, cell motility and intracellular vesicle transport. As Myo21 contains two ubiquitin associated (UBA)-like domains (UBLD) in its amino acid sequence, we considered it of interest to analyze the role of these domains in the intracellular distribution and functions of this protein in Leishmania cells. In this context, we created green fluorescent protein (GFP)-conjugates of Myo21 constructs lacking one of the two UBLDs at a time or both the UBLDs as well as GFP-conjugates of only the two UBLDs and Myo21 tail lacking the two UBLDs and separately expressed them in the Leishmania cells. Our results show that unlike Myo21-GFP, Myo21-GFP constructs lacking either one or both the UBLDs failed to concentrate and co-distribute with actin in the proximal region of the flagellum. Nevertheless, the GFP conjugate of only the two UBLDs was found to predominantly localize to the flagellum base. Additionally, the cells that expressed only one or both the UBLDs-deleted Myo21-GFP constructs possessed shorter flagellum and displayed slower motility, compared to Myo21-GFP expressing cells. Further, the intracellular vesicle transport and cell growth were severely impaired in the cells that expressed both the UBLDs deleted Myo21-GFP construct, but in contrast, virtually no effect was observed on the intracellular vesicle transport and growth in the cells that expressed single UBLD deleted mutant proteins. Moreover, the observed slower growth of both the UBLDs-deleted Myo21-GFP expressing cells was primarily due to delayed G2/M phase caused by aberrant nuclear and daughter cell segregation during their cell division process. These results taken together clearly reveal that the presence of UBLDs in Myo21 are essentially required for its predominant localization to the flagellum base, and perhaps also in its involvement in the flagellum assembly and cell division. Possible role of UBLDs in involvement of Myo21 during Leishmania flagellum assembly and cell cycle is discussed.

Summary: Twinfilin is an evolutionarily conserved actin‐binding protein, which regulates actin‐dynamics in eukaryotic cells. Homologs of this protein have been detected in the genome of various protozoan parasites causing diseases in human. However, very little is known about their core functions in these organisms. We show here that a twinfilin homolog in a human pathogen Leishmania, primarily localizes to the nucleolus and, to some extent, also in the basal body region. In the dividing cells, nucleolar twinfilin redistributes to the mitotic spindle and remains there partly associated with the spindle microtubules. We further show that approximately 50% depletion of this protein significantly retards the cell growth due to sluggish progression of S phase of the cell division cycle, owing to the delayed nuclear DNA synthesis. Interestingly, overexpression of this protein results in significantly increased length of the mitotic spindle in the dividing Leishmania cells, whereas, its depletion adversely affects spindle elongation and architecture. Our results indicate that twinfilin controls on one hand, the DNA synthesis and on the other, the mitotic spindle elongation, thus contributing to karyokinesis in Leishmania.

Coronin proteins bind with actin filaments and participate in regulation of actin‐dependent processes. These proteins contain a coiled‐coil domain at their C‐terminus, which is responsible for their dimeric or trimeric forms. However, the functional significance of these oligomeric configurations in organizing the actin cytoskeleton is obscure. Here, we report that the Leishmania coronin exists in a higher oligomeric form through its coiled‐coil domain, the truncation of which ablates the ability of Leishmania coronin to assist actin‐filament formation. F‐actin co‐sedimentation assay using purified proteins shows that the coiled‐coil domain does not interact with actin‐filaments and its absence does not abrogate actin–coronin interaction. Furthermore, it was shown that unlike other coronins, Leishmania coronin interacts with actin‐filaments through its unique region. These results provided important insights into the role of coronin oligomerization in modulating actin‐network.