Biology subjects

Myskova, J.

Publications and source records attributed to Myskova, J..

3 recordsLinked to original sources

bioRxiv2025

Directionality of two-photon absorption in representative fluorescent proteins

Molecules of fluorescent proteins (FPs) exhibit anisotropic optical properties. While the directionality of single-photon (1P) absorption and emission in the most commonly used FPs has already been characterized, directionality of their two-photon (2P) excitation remains poorly understood, even though it offers higher sensitivity to molecular orientation and promises to yield more information on molecular orientational distributions than the 1P processes. By measuring optical properties of FP crystals, we have now determined the two-photon absorptivity tensors (2PATs) of the FPs mTurquoise2, eGFP and mCherry. Our results demonstrate the feasibility of experimental 2PAT determinations, provide new insights into molecular mechanisms of 2P absorption in FPs, open a new avenue to rational development of novel biosensors and allow detailed structural interpretation of results obtained with such biosensors.

biophysics↗

bioRxiv2020

Role for the flagellum attachment zone in the resolution of cell membrane morphogenesis during Leishmania cell division

The shape and form of the flagellated eukaryotic parasite Leishmania is sculpted to its ecological niches and needs to be transmitted to each generation with great fidelity. The shape of the Leishmania cell is defined by the sub-pellicular microtubule array and the positioning of the nucleus, kinetoplast and the flagellum within this array. The flagellum emerges from the anterior end of the cell body through an invagination of the cell body membrane called the flagellar pocket. Within the flagellar pocket the flagellum is laterally attached to the side of the flagellar pocket by a cytoskeletal structure called the flagellum attachment zone (FAZ). During the cell cycle single copy organelles duplicate with a new flagellum assembling alongside the old flagellum and these are then segregated between the two daughter cells by cytokinesis, which initiates at the anterior cell tip. Here, we have investigated the role of the FAZ in the morphogenetic resolution of the anterior cell tip during cell division. We have deleted the FAZ filament protein, FAZ2 and investigated its function using light and electron microscopy and infection studies. The loss of FAZ2 caused a disruption in membrane organisation at the anterior cell tip, resulting in cells that late in division were connected to each other by a membranous bridge structure between their flagella. These changes had a great impact in vivo with the FAZ2 null mutant unable to develop and proliferate in sand flies and causing a reduced parasite burden in mice. Our study provides a deeper understanding of membrane-cytoskeletal interactions that define the shape and form of an individual cell and the remodelling of that form during cell division. Author summaryLeishmania are parasites that cause leishmaniasis in humans with symptoms ranging from mild cutaneous lesions to severe visceral disease. The life cycle of these parasites alternates between the human host and the sand fly vector, with distinct forms in both. These different forms have different cell shapes that are adapted for survival in these different environments. Leishmania parasites have an elongated cell shape with a flagellum extending from one end and this shape is due to a protein skeleton beneath the cell membrane. This skeleton is made up of different units one of which is called the flagellum attachment zone (FAZ), that connects the flagellum to the cell body. We have found that one of the proteins in the FAZ called FAZ2 is important for generating the shape of the cell at the point where the flagellum exits the cell. When we deleted FAZ2 we found that the cell membrane at the tip of the was distorted resulting in unusual connections between the flagella of different cells. We found that the disruption to the cell shape reduces the ability of the parasite to infect mice and develop in the sand fly, which shows the importance of the parasite shape.

cell biology↗

bioRxiv2018

Genetic dissection of a Leishmania flagellar proteome demonstrates requirement for directional motility in sand fly infections

The protozoan parasite Leishmania possesses a single flagellum, which is remodelled during the parasites life cycle from a long motile flagellum in promastigote forms in the sand fly to a short immotile flagellum in amastigotes residing in mammalian phagocytes. This study examined the protein composition and in vivo function of the promastigote flagellum. Protein mass spectrometry and label free protein enrichment testing of isolated flagella and deflagellated cell bodies defined a flagellar proteome for L. mexicana promastigote forms (available via ProteomeXchange with identifier PXD011057). This information was used to generate a CRISPR-Cas9 knockout library of 100 mutants to screen for flagellar defects. This first large-scale knockout screen in a Leishmania sp. identified 56 mutants with altered swimming speed (52 reduced and 4 increased) and defined distinct mutant categories (faster swimmers, slower swimmers, slow uncoordinated swimmers and paralysed cells, including aflagellate promastigotes and cells with curled flagella and disruptions of the paraflagellar rod). Each mutant was tagged with a unique 17-nt barcode, providing a simple barcode sequencing (bar-seq) method for measuring the relative fitness of L. mexicana mutants in vivo. In mixed infections of the permissive sand fly vector Lutzomyia longipalpis, paralysed promastigotes and uncoordinated swimmers were severely diminished in the fly after defecation of the bloodmeal. Subsequent examination of flies infected with a single mutant lacking the central pair protein PF16 showed that these paralysed promastigotes did not reach anterior regions of the fly alimentary tract. These data show that L. mexicana need directional motility for successful colonisation of sand flies.\n\nAuthor SummaryLeishmania are protozoan parasites, transmitted between mammals by the bite of phlebotomine sand flies. Promastigote forms in the sand fly have a long flagellum, which is motile and used for anchoring the parasites to prevent clearance with the digested blood meal remnants. To dissect flagellar functions and their importance in life cycle progression, we generated here a comprehensive list of >300 flagellar proteins and produced a CRISPR-Cas9 gene knockout library of 100 mutant Leishmania. We studied their behaviour in vitro before examining their fate in the sand fly Lutzomyia longipalpis. Measuring mutant swimming speeds showed that about half behaved differently compared to the wild type: a few swam faster, many slower and some were completely paralysed. We also found a group of uncoordinated swimmers. To test whether flagellar motility is required for parasite migration from the fly midgut to the foregut from where they reach the next host, we infected sand flies with a mixed mutant population. Each mutant carried a unique tag and tracking these tags up to nine days after infection showed that paralysed and uncoordinated Leishmania were rapidly lost from flies. These data indicate that directional swimming is important for successful colonisation of sand flies.

cell biology↗