József Mihály - Profile on Academia.edu (original) (raw)
Papers by József Mihály
Journal of Cell Science, Jun 5, 2023
Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin... more Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin filaments. The actin and myosin filaments are organized into repeated units called sarcomeres, which form the myofibrils. Muscle contraction is achieved by the simultaneous shortening of sarcomeres, which requires all sarcomeres to be the same size. Muscles have a variety of ways to ensure sarcomere homogeneity. We previously showed that the controlled oligomerization of Zasp proteins sets the diameter of the myofibril. Here we looked for Zaspbinding proteins at the Z-disc to identify additional proteins coordinating myofibril growth and assembly. We found that the E1 subunit of the oxoglutarate dehydrogenase complex localizes to both the Z-disc and the mitochondria, and is recruited to the Z-disc by Zasp52. The three subunits of the oxoglutarate dehydrogenase complex are required for myofibril formation. Using super-resolution microscopy, we revealed the overall organization of the complex at the Z-disc. Metabolomics identified an amino acid imbalance affecting protein synthesis as a possible cause of myofibril defects, which is supported by OGDH-dependent localization of ribosomes at the Z-disc.
Bio-protocol, 2020
Sarcomeres are extremely highly ordered macromolecular assemblies where proper structural organiz... more Sarcomeres are extremely highly ordered macromolecular assemblies where proper structural organization is an absolute prerequisite to the functionality of these contractile units. Despite the wealth of information collected, the exact spatial arrangement of many of the H-zone and Z-disk proteins remained unknown. Recently, we developed a powerful nanoscopic approach to localize the sarcomeric protein components with a resolution well below the diffraction limit. The ease of sample preparation and the near crystalline structure of the Drosophila flight muscle sarcomeres make them ideally suitable for single molecule localization microscopy and structure averaging. Our approach allowed us to determine the position of dozens of H-zone and Z-disk proteins with a quasi-molecular, ~5-10 nm localization precision. The protocol described below provides an easy and reproducible method to prepare individual myofibrils for dSTORM imaging. In addition, it includes an in-depth description of a custom made and freely available software toolbox to process and quantitatively analyze the raw localization data.
Concurrent activating mutations of the Notch and PI3K/Akt signalling pathways cooperate in the in... more Concurrent activating mutations of the Notch and PI3K/Akt signalling pathways cooperate in the induction of aggressive cancers. Unfortunately, direct targeting of any of these aberrant pathways can result in severe side effects due to their broad physiological roles in multiple organs. Here, using an unbiased chemical in vivo screen in Drosophila we identified compounds that suppress the activity of the pro-inflammatory enzymes, nitric oxide synthase (NOS) and lipoxygenase (LOX), capable to block oncogenic Notch-PI3K/Akt cooperation without unwanted side effects. Genetic inactivation of NOS and LOX signalling components mirrors the anti-tumorigenic effect of the hit compounds. We show that NOS activity and immunosuppression associated to inflammation facilitates Notch-mediated tumorigenesis. Our study reveals an unnoticed immune inflammatory process underlying Notch-PI3K/Akt tumours and exposes NOS as a druggable target for anti-cancer therapeutic development.
Neural Regeneration Research, 2017
It is a nice and short reviewer article. This article summarizes the most recent advancements reg... more It is a nice and short reviewer article. This article summarizes the most recent advancements regarding the role(s) played by formin in joining the actin and microtubule filaments in axonal growth cones. In addition, the author also point out several possible lines of related research in the future, including the studies of how formins interact with +TIP proteins and of how formin/+TIP complex contributes to linking the actin and microtubule filaments in growth cones. The findings of several recent and relevant articles relating to the above-described subject have been discussed in the article. This article also includes a good background introduction which helps guide readers from different fields to read through the article.
Scientific Reports, Jan 28, 2023
Object detection is an image analysis task with a wide range of applications, which is difficult ... more Object detection is an image analysis task with a wide range of applications, which is difficult to accomplish with traditional programming. Recent breakthroughs in machine learning have made significant progress in this area. However, these algorithms are generally compatible with traditional pixelated images and cannot be directly applied for pointillist datasets generated by single molecule localization microscopy (SMLM) methods. Here, we have improved the averaging method developed for the analysis of SMLM images of sarcomere structures based on a machine learning object detection algorithm. The ordered structure of sarcomeres allows us to determine the location of the proteins more accurately by superimposing SMLM images of identically assembled proteins. However, the area segmentation process required for averaging can be extremely time-consuming and tedious. In this work, we have automated this process. The developed algorithm not only finds the regions of interest, but also classifies the localizations and identifies the true positive ones. For training, we used simulations to generate large amounts of labelled data. After tuning the neural network's internal parameters, it could find the localizations associated with the structures we were looking for with high accuracy. We validated our results by comparing them with previous manual evaluations. It has also been proven that the simulations can generate data of sufficient quality for training. Our method is suitable for the identification of other types of structures in SMLM data. Single molecule localization microscopy (SMLM) 1-4 has become a widely used and accepted tool in molecular cell biology research . By utilizing the localization of single molecules, previously unseen spatial resolution ( ∼ 10 nm) has been achieved in the optical regime 6 . The raw data provided by SMLM is a point cloud, i.e. a list of spatial coordinates of the localized emitters, which is fundamentally different from the pixelated images of conventional optical microscopes. Consequently, the interpretation, quantification and visualization of such data require new approaches and solutions. Conventional pixelated images can be generated from the localization data , however such conversion introduces a loss of information . Therefore, the direct extraction of the relevant information from the raw localization data requires extra effort. Another hurdle of the interpretation of SMLM measurements is its labour intensity. Data evaluation often requires the analysis of data belonging to many identical structures. Selecting the structures of interest and analyzing them individually is time consuming and tedious if performed manually. To this end, object classification 15 and structure averaging 16 methods have been developed and made public recently. Machine learning algorithms are gaining widespread attention for the analysis of complex data 17 . Sometimes, it is difficult to write an exact algorithm that the computer can follow to solve a specific task. In such cases, one option is to use machine learning methods. If we know the possible response signal of the system for a given input, supervised machine learning can be applied. Otherwise, without prior knowledge, non-supervised machine learning algorithms can be used to find patterns in the data or label data points. Artificial Neural Networks (ANNs) are widely used in supervised machine learning. They are made up of artificial neurons that can receive and process input data, and subsequently provide an output. Neurons with similar functions are grouped together to form layers. The machine tunes the internal parameters of the neural network based on a known training dataset so that its output converges to the expected output. The data generated by localization algorithms are not directly compatible with most neural network constructs used for
Journal of Cell Science
Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin... more Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin filaments. The actin and myosin filaments are organized into repeated units called sarcomeres, which form the myofibrils. Muscle contraction is achieved by the simultaneous shortening of sarcomeres, which requires all sarcomeres to be the same size. Muscles have a variety of ways to ensure sarcomere homogeneity. We have previously shown that the controlled oligomerization of Zasp proteins sets the diameter of the myofibril. Here, we looked for Zasp-binding proteins at the Z-disc to identify additional proteins coordinating myofibril growth and assembly. We found that the E1 subunit of the oxoglutarate dehydrogenase complex localizes to both the Z-disc and the mitochondria, and is recruited to the Z-disc by Zasp52. The three subunits of the oxoglutarate dehydrogenase complex are required for myofibril formation. Using super-resolution microscopy, we revealed the overall organization of t...
Resolution and Discovery
Localization based superresolution technique provides the highest spatial resolution in optical m... more Localization based superresolution technique provides the highest spatial resolution in optical microscopy. The final image is formed by the precise localization of individual fluorescent dyes, therefore the quantification of the collected data requires special protocols, algorithms and validation processes. The effects of labelling density and structured background on the final image quality were studied theoretically using the TestSTORM simulator. It was shown that system parameters affect the morphology of the final reconstructed image in different ways and the accuracy of the imaging can be determined. Although theoretical studies help in the optimization procedure, the quantification of experimental data raises additional issues, since the ground truth data is unknown. Localization precision, linker length, sample drift and labelling density are the major factors that make quantitative data analysis difficult. Two examples (geometrical evaluation of sarcomere structures and cou...
PLOS Genetics, Jan 9, 2024
During striated muscle development the first periodically repeated units appear in the premyofibr... more During striated muscle development the first periodically repeated units appear in the premyofibrils, consisting of immature sarcomeres that must undergo a substantial growth both in length and width, to reach their final size. Here we report that, beyond its well established role in sarcomere elongation, the Sarcomere length short (SALS) protein is involved in Z-disc formation and peripheral growth of the sarcomeres. Our protein localization data and loss-offunction studies in the Drosophila indirect flight muscle strongly suggest that radial growth of the sarcomeres is initiated at the Z-disc. As to thin filament elongation, we used a powerful nanoscopy approach to reveal that SALS is subject to a major conformational change during sarcomere development, which might be critical to stop pointed end elongation in the adult muscles. In addition, we demonstrate that the roles of SALS in sarcomere elongation and radial growth are both dependent on formin type of actin assembly factors. Unexpectedly, when SALS is present in excess amounts, it promotes the formation of actin aggregates highly resembling the ones described in nemaline myopathy patients. Collectively, these findings helped to shed light on the complex mechanisms of SALS during the coordinated elongation and thickening of the sarcomeres, and resulted in the discovery of a potential nemaline myopathy model, suitable for the identification of genetic and small molecule inhibitors.
Development
Optical insulation of the unit eyes (ommatidia) is an important prerequisite of precise sight wit... more Optical insulation of the unit eyes (ommatidia) is an important prerequisite of precise sight with compound eyes. Separation of the ommatidia is ensured by pigment cells that organize into a hexagonal lattice in the Drosophila eye, forming thin walls between the facets. Cell adhesion, mediated by apically and latero-basally located junctional complexes, is crucial for stable attachment of these cells to each other and the basal lamina. Whereas former studies have focused on the formation and remodelling of the cellular connections at the apical region, here, we report a specific alteration of the lateral adhesion of the lattice cells, leaving the apical junctions largely unaffected. We found that DAAM and FRL, two formin-type cytoskeleton regulatory proteins, play redundant roles in lateral adhesion of the interommatidial cells and patterning of the retinal floor. We show that formin-dependent cortical actin assembly is crucial for latero-basal sealing of the ommatidial lattice. We ...
Cells, May 4, 2022
Dorsal closure is a late embryogenesis process required to seal the epidermal hole on the dorsal ... more Dorsal closure is a late embryogenesis process required to seal the epidermal hole on the dorsal side of the Drosophila embryo. This process involves the coordination of several forces generated in the epidermal cell layer and in the amnioserosa cells, covering the hole. Ultimately, these forces arise due to cytoskeletal rearrangements that induce changes in cell shape and result in tissue movement. While a number of cytoskeleton regulatory proteins have already been linked to dorsal closure, here we expand this list by demonstrating that four of the six Drosophila formin type actin assembly factors are needed to bring about the proper fusion of the epithelia. An analysis of the morphological and dynamic properties of dorsal closure in formin mutants revealed a differential contribution for each formin, although we found evidence for functional redundancies as well. Therefore, we propose that the four formins promote the formation of several, and only partly identical, actin structures each with a specific role in the mechanics of dorsal closure.
Cells, Jul 29, 2021
With the advent of super-resolution microscopy, we gained a powerful toolbox to bridge the gap be... more With the advent of super-resolution microscopy, we gained a powerful toolbox to bridge the gap between the cellular-and molecular-level analysis of living organisms. Although nanoscopy is broadly applicable, classical model organisms, such as fruit flies, worms and mice, remained the leading subjects because combining the strength of sophisticated genetics, biochemistry and electrophysiology with the unparalleled resolution provided by super-resolution imaging appears as one of the most efficient approaches to understanding the basic cell biological questions and the molecular complexity of life. Here, we summarize the major nanoscopic techniques and illustrate how these approaches were used in Drosophila model systems to revisit a series of well-known cell biological phenomena. These investigations clearly demonstrate that instead of simply achieving an improvement in image quality, nanoscopy goes far beyond with its immense potential to discover novel structural and mechanistic aspects. With the examples of synaptic active zones, centrosomes and sarcomeres, we will explain the instrumental role of super-resolution imaging pioneered in Drosophila in understanding fundamental subcellular constituents.
International Journal of Molecular Sciences, May 10, 2022
The actin containing tropomyosin and troponin decorated thin filaments form one of the crucial co... more The actin containing tropomyosin and troponin decorated thin filaments form one of the crucial components of the contractile apparatus in muscles. The thin filaments are organized into densely packed lattices interdigitated with myosin-based thick filaments. The crossbridge interactions between these myofilaments drive muscle contraction, and the degree of myofilament overlap is a key factor of contractile force determination. As such, the optimal length of the thin filaments is critical for efficient activity, therefore, this parameter is precisely controlled according to the workload of a given muscle. Thin filament length is thought to be regulated by two major, but only partially understood mechanisms: it is set by (i) factors that mediate the assembly of filaments from monomers and catalyze their elongation, and (ii) by factors that specify their length and uniformity. Mutations affecting these factors can alter the length of thin filaments, and in human cases, many of them are linked to debilitating diseases such as nemaline myopathy and dilated cardiomyopathy.
Genetics, 2016
The noncanonical Frizzled/planar cell polarity (PCP) pathway regulates establishment of polarity ... more The noncanonical Frizzled/planar cell polarity (PCP) pathway regulates establishment of polarity within the plane of an epithelium to generate diversity of cell fates, asymmetric, but highly aligned structures, or to orchestrate the directional migration of cells during convergent extension during vertebrate gastrulation. In Drosophila, PCP signaling is essential to orient actin wing hairs and to align ommatidia in the eye, in part by coordinating the movement of groups of photoreceptor cells during ommatidial rotation. Importantly, the coordination of PCP signaling with changes in the cytoskeleton is essential for proper epithelial polarity. Formins polymerize linear actin filaments and are key regulators of the actin cytoskeleton. Here, we show that the diaphanous-related formin, Frl, the single fly member of the FMNL (formin related in leukocytes/formin-like) formin subfamily affects ommatidial rotation in the Drosophila eye and is controlled by the Rho family GTPase Cdc42. Inter...
bioRxiv, 2021
Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin... more Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin filaments. The actin and myosin filaments are organized into repeated units called sarcomeres, which form the myofibril cables. Muscle contraction is achieved by the simultaneous shortening of sarcomeres and for a highly coordinated contraction to occur all sarcomeres should have the same size. Muscles have evolved a variety of ways to ensure sarcomere homogeneity, one example being the controlled oligomerization of Zasp proteins that sets the diameter of the myofibril. To understand how Zasp proteins effect myofibril growth, we looked for Zasp-binding proteins at the Z-disc. We found that the E1 subunit of the oxoglutarate dehydrogenase complex is recruited to the Z-disc by Zasp52 and is required to sustain myofibril growth. By making specific mutants, we show that its enzymatic activity is important for myofibril growth, and that the other two subunits of the complex are also required...
Genetics, 2000
The Drosophila melanogaster Ketel gene was identified via the KetelD dominant female sterile muta... more The Drosophila melanogaster Ketel gene was identified via the KetelD dominant female sterile mutations and their ketelr revertant alleles that are recessive zygotic lethals. The maternally acting KetelD mutations inhibit cleavage nuclei formation. We cloned the Ketel gene on the basis of a common breakpoint in 38E1.2-3 in four ketelr alleles. The Ketel+ transgenes rescue ketelr-associated zygotic lethality and slightly reduce KetelD-associated dominant female sterility. Ketel is a single copy gene. It is transcribed to a single 3.6-kb mRNA, predicted to encode the 97-kD Ketel protein. The 884-amino-acid sequence of Ketel is 60% identical and 78% similar to that of human importin-β, the nuclear import receptor for proteins with a classical NLS. Indeed, Ketel supports import of appropriately designed substrates into nuclei of digitonin-permeabilized HeLa cells. As shown by a polyclonal anti-Ketel antibody, nurse cells synthesize and transfer Ketel protein into the oocyte cytoplasm fro...
International Journal of Molecular Sciences
The actin containing tropomyosin and troponin decorated thin filaments form one of the crucial co... more The actin containing tropomyosin and troponin decorated thin filaments form one of the crucial components of the contractile apparatus in muscles. The thin filaments are organized into densely packed lattices interdigitated with myosin-based thick filaments. The crossbridge interactions between these myofilaments drive muscle contraction, and the degree of myofilament overlap is a key factor of contractile force determination. As such, the optimal length of the thin filaments is critical for efficient activity, therefore, this parameter is precisely controlled according to the workload of a given muscle. Thin filament length is thought to be regulated by two major, but only partially understood mechanisms: it is set by (i) factors that mediate the assembly of filaments from monomers and catalyze their elongation, and (ii) by factors that specify their length and uniformity. Mutations affecting these factors can alter the length of thin filaments, and in human cases, many of them are...
Applied Microbiology and Biotechnology, 2005
Biochemical and pharmacological properties of biosurfactants produced at 45°C temperature by Pseu... more Biochemical and pharmacological properties of biosurfactants produced at 45°C temperature by Pseudomonas aeruginosa mucoid (M) and non-mucoid (NM) strains, isolated from hydrocarbon-contaminated soil samples, were characterized. Both the strains secreted appreciable amount of biosurfactants (5.0-6.5 g/l), responsible for the reduction of surface tension of the medium from 68 to 29±0.5 mN/m post 96 h of growth. Maximum yield of biosurfactants was observed following the supplementation of NH 4 Cl and glycerol as nitrogenous source and carbon source, respectively. These thermostable biosurfactants exhibited strong emulsifying property and could release appreciable amount of oil from saturated sand-pack column. Pharmacological characterization of these biosurfactants revealed that they induced dose-dependent hemolysis and coagulation of platelet-poor plasma but were non-detrimental to chicken lung, liver, heart and kidney tissues. Our study has documented that biosurfactants from P. aeruginosa M and NM strains could be exploited for use in petroleum sectors as well in pharmaceutical industries.
PLoS Genetics, 2014
During muscle development, myosin and actin containing filaments assemble into the highly organiz... more During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Zdisc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin.
Journal of Cell Science, Jun 5, 2023
Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin... more Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin filaments. The actin and myosin filaments are organized into repeated units called sarcomeres, which form the myofibrils. Muscle contraction is achieved by the simultaneous shortening of sarcomeres, which requires all sarcomeres to be the same size. Muscles have a variety of ways to ensure sarcomere homogeneity. We previously showed that the controlled oligomerization of Zasp proteins sets the diameter of the myofibril. Here we looked for Zaspbinding proteins at the Z-disc to identify additional proteins coordinating myofibril growth and assembly. We found that the E1 subunit of the oxoglutarate dehydrogenase complex localizes to both the Z-disc and the mitochondria, and is recruited to the Z-disc by Zasp52. The three subunits of the oxoglutarate dehydrogenase complex are required for myofibril formation. Using super-resolution microscopy, we revealed the overall organization of the complex at the Z-disc. Metabolomics identified an amino acid imbalance affecting protein synthesis as a possible cause of myofibril defects, which is supported by OGDH-dependent localization of ribosomes at the Z-disc.
Bio-protocol, 2020
Sarcomeres are extremely highly ordered macromolecular assemblies where proper structural organiz... more Sarcomeres are extremely highly ordered macromolecular assemblies where proper structural organization is an absolute prerequisite to the functionality of these contractile units. Despite the wealth of information collected, the exact spatial arrangement of many of the H-zone and Z-disk proteins remained unknown. Recently, we developed a powerful nanoscopic approach to localize the sarcomeric protein components with a resolution well below the diffraction limit. The ease of sample preparation and the near crystalline structure of the Drosophila flight muscle sarcomeres make them ideally suitable for single molecule localization microscopy and structure averaging. Our approach allowed us to determine the position of dozens of H-zone and Z-disk proteins with a quasi-molecular, ~5-10 nm localization precision. The protocol described below provides an easy and reproducible method to prepare individual myofibrils for dSTORM imaging. In addition, it includes an in-depth description of a custom made and freely available software toolbox to process and quantitatively analyze the raw localization data.
Concurrent activating mutations of the Notch and PI3K/Akt signalling pathways cooperate in the in... more Concurrent activating mutations of the Notch and PI3K/Akt signalling pathways cooperate in the induction of aggressive cancers. Unfortunately, direct targeting of any of these aberrant pathways can result in severe side effects due to their broad physiological roles in multiple organs. Here, using an unbiased chemical in vivo screen in Drosophila we identified compounds that suppress the activity of the pro-inflammatory enzymes, nitric oxide synthase (NOS) and lipoxygenase (LOX), capable to block oncogenic Notch-PI3K/Akt cooperation without unwanted side effects. Genetic inactivation of NOS and LOX signalling components mirrors the anti-tumorigenic effect of the hit compounds. We show that NOS activity and immunosuppression associated to inflammation facilitates Notch-mediated tumorigenesis. Our study reveals an unnoticed immune inflammatory process underlying Notch-PI3K/Akt tumours and exposes NOS as a druggable target for anti-cancer therapeutic development.
Neural Regeneration Research, 2017
It is a nice and short reviewer article. This article summarizes the most recent advancements reg... more It is a nice and short reviewer article. This article summarizes the most recent advancements regarding the role(s) played by formin in joining the actin and microtubule filaments in axonal growth cones. In addition, the author also point out several possible lines of related research in the future, including the studies of how formins interact with +TIP proteins and of how formin/+TIP complex contributes to linking the actin and microtubule filaments in growth cones. The findings of several recent and relevant articles relating to the above-described subject have been discussed in the article. This article also includes a good background introduction which helps guide readers from different fields to read through the article.
Scientific Reports, Jan 28, 2023
Object detection is an image analysis task with a wide range of applications, which is difficult ... more Object detection is an image analysis task with a wide range of applications, which is difficult to accomplish with traditional programming. Recent breakthroughs in machine learning have made significant progress in this area. However, these algorithms are generally compatible with traditional pixelated images and cannot be directly applied for pointillist datasets generated by single molecule localization microscopy (SMLM) methods. Here, we have improved the averaging method developed for the analysis of SMLM images of sarcomere structures based on a machine learning object detection algorithm. The ordered structure of sarcomeres allows us to determine the location of the proteins more accurately by superimposing SMLM images of identically assembled proteins. However, the area segmentation process required for averaging can be extremely time-consuming and tedious. In this work, we have automated this process. The developed algorithm not only finds the regions of interest, but also classifies the localizations and identifies the true positive ones. For training, we used simulations to generate large amounts of labelled data. After tuning the neural network's internal parameters, it could find the localizations associated with the structures we were looking for with high accuracy. We validated our results by comparing them with previous manual evaluations. It has also been proven that the simulations can generate data of sufficient quality for training. Our method is suitable for the identification of other types of structures in SMLM data. Single molecule localization microscopy (SMLM) 1-4 has become a widely used and accepted tool in molecular cell biology research . By utilizing the localization of single molecules, previously unseen spatial resolution ( ∼ 10 nm) has been achieved in the optical regime 6 . The raw data provided by SMLM is a point cloud, i.e. a list of spatial coordinates of the localized emitters, which is fundamentally different from the pixelated images of conventional optical microscopes. Consequently, the interpretation, quantification and visualization of such data require new approaches and solutions. Conventional pixelated images can be generated from the localization data , however such conversion introduces a loss of information . Therefore, the direct extraction of the relevant information from the raw localization data requires extra effort. Another hurdle of the interpretation of SMLM measurements is its labour intensity. Data evaluation often requires the analysis of data belonging to many identical structures. Selecting the structures of interest and analyzing them individually is time consuming and tedious if performed manually. To this end, object classification 15 and structure averaging 16 methods have been developed and made public recently. Machine learning algorithms are gaining widespread attention for the analysis of complex data 17 . Sometimes, it is difficult to write an exact algorithm that the computer can follow to solve a specific task. In such cases, one option is to use machine learning methods. If we know the possible response signal of the system for a given input, supervised machine learning can be applied. Otherwise, without prior knowledge, non-supervised machine learning algorithms can be used to find patterns in the data or label data points. Artificial Neural Networks (ANNs) are widely used in supervised machine learning. They are made up of artificial neurons that can receive and process input data, and subsequently provide an output. Neurons with similar functions are grouped together to form layers. The machine tunes the internal parameters of the neural network based on a known training dataset so that its output converges to the expected output. The data generated by localization algorithms are not directly compatible with most neural network constructs used for
Journal of Cell Science
Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin... more Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin filaments. The actin and myosin filaments are organized into repeated units called sarcomeres, which form the myofibrils. Muscle contraction is achieved by the simultaneous shortening of sarcomeres, which requires all sarcomeres to be the same size. Muscles have a variety of ways to ensure sarcomere homogeneity. We have previously shown that the controlled oligomerization of Zasp proteins sets the diameter of the myofibril. Here, we looked for Zasp-binding proteins at the Z-disc to identify additional proteins coordinating myofibril growth and assembly. We found that the E1 subunit of the oxoglutarate dehydrogenase complex localizes to both the Z-disc and the mitochondria, and is recruited to the Z-disc by Zasp52. The three subunits of the oxoglutarate dehydrogenase complex are required for myofibril formation. Using super-resolution microscopy, we revealed the overall organization of t...
Resolution and Discovery
Localization based superresolution technique provides the highest spatial resolution in optical m... more Localization based superresolution technique provides the highest spatial resolution in optical microscopy. The final image is formed by the precise localization of individual fluorescent dyes, therefore the quantification of the collected data requires special protocols, algorithms and validation processes. The effects of labelling density and structured background on the final image quality were studied theoretically using the TestSTORM simulator. It was shown that system parameters affect the morphology of the final reconstructed image in different ways and the accuracy of the imaging can be determined. Although theoretical studies help in the optimization procedure, the quantification of experimental data raises additional issues, since the ground truth data is unknown. Localization precision, linker length, sample drift and labelling density are the major factors that make quantitative data analysis difficult. Two examples (geometrical evaluation of sarcomere structures and cou...
PLOS Genetics, Jan 9, 2024
During striated muscle development the first periodically repeated units appear in the premyofibr... more During striated muscle development the first periodically repeated units appear in the premyofibrils, consisting of immature sarcomeres that must undergo a substantial growth both in length and width, to reach their final size. Here we report that, beyond its well established role in sarcomere elongation, the Sarcomere length short (SALS) protein is involved in Z-disc formation and peripheral growth of the sarcomeres. Our protein localization data and loss-offunction studies in the Drosophila indirect flight muscle strongly suggest that radial growth of the sarcomeres is initiated at the Z-disc. As to thin filament elongation, we used a powerful nanoscopy approach to reveal that SALS is subject to a major conformational change during sarcomere development, which might be critical to stop pointed end elongation in the adult muscles. In addition, we demonstrate that the roles of SALS in sarcomere elongation and radial growth are both dependent on formin type of actin assembly factors. Unexpectedly, when SALS is present in excess amounts, it promotes the formation of actin aggregates highly resembling the ones described in nemaline myopathy patients. Collectively, these findings helped to shed light on the complex mechanisms of SALS during the coordinated elongation and thickening of the sarcomeres, and resulted in the discovery of a potential nemaline myopathy model, suitable for the identification of genetic and small molecule inhibitors.
Development
Optical insulation of the unit eyes (ommatidia) is an important prerequisite of precise sight wit... more Optical insulation of the unit eyes (ommatidia) is an important prerequisite of precise sight with compound eyes. Separation of the ommatidia is ensured by pigment cells that organize into a hexagonal lattice in the Drosophila eye, forming thin walls between the facets. Cell adhesion, mediated by apically and latero-basally located junctional complexes, is crucial for stable attachment of these cells to each other and the basal lamina. Whereas former studies have focused on the formation and remodelling of the cellular connections at the apical region, here, we report a specific alteration of the lateral adhesion of the lattice cells, leaving the apical junctions largely unaffected. We found that DAAM and FRL, two formin-type cytoskeleton regulatory proteins, play redundant roles in lateral adhesion of the interommatidial cells and patterning of the retinal floor. We show that formin-dependent cortical actin assembly is crucial for latero-basal sealing of the ommatidial lattice. We ...
Cells, May 4, 2022
Dorsal closure is a late embryogenesis process required to seal the epidermal hole on the dorsal ... more Dorsal closure is a late embryogenesis process required to seal the epidermal hole on the dorsal side of the Drosophila embryo. This process involves the coordination of several forces generated in the epidermal cell layer and in the amnioserosa cells, covering the hole. Ultimately, these forces arise due to cytoskeletal rearrangements that induce changes in cell shape and result in tissue movement. While a number of cytoskeleton regulatory proteins have already been linked to dorsal closure, here we expand this list by demonstrating that four of the six Drosophila formin type actin assembly factors are needed to bring about the proper fusion of the epithelia. An analysis of the morphological and dynamic properties of dorsal closure in formin mutants revealed a differential contribution for each formin, although we found evidence for functional redundancies as well. Therefore, we propose that the four formins promote the formation of several, and only partly identical, actin structures each with a specific role in the mechanics of dorsal closure.
Cells, Jul 29, 2021
With the advent of super-resolution microscopy, we gained a powerful toolbox to bridge the gap be... more With the advent of super-resolution microscopy, we gained a powerful toolbox to bridge the gap between the cellular-and molecular-level analysis of living organisms. Although nanoscopy is broadly applicable, classical model organisms, such as fruit flies, worms and mice, remained the leading subjects because combining the strength of sophisticated genetics, biochemistry and electrophysiology with the unparalleled resolution provided by super-resolution imaging appears as one of the most efficient approaches to understanding the basic cell biological questions and the molecular complexity of life. Here, we summarize the major nanoscopic techniques and illustrate how these approaches were used in Drosophila model systems to revisit a series of well-known cell biological phenomena. These investigations clearly demonstrate that instead of simply achieving an improvement in image quality, nanoscopy goes far beyond with its immense potential to discover novel structural and mechanistic aspects. With the examples of synaptic active zones, centrosomes and sarcomeres, we will explain the instrumental role of super-resolution imaging pioneered in Drosophila in understanding fundamental subcellular constituents.
International Journal of Molecular Sciences, May 10, 2022
The actin containing tropomyosin and troponin decorated thin filaments form one of the crucial co... more The actin containing tropomyosin and troponin decorated thin filaments form one of the crucial components of the contractile apparatus in muscles. The thin filaments are organized into densely packed lattices interdigitated with myosin-based thick filaments. The crossbridge interactions between these myofilaments drive muscle contraction, and the degree of myofilament overlap is a key factor of contractile force determination. As such, the optimal length of the thin filaments is critical for efficient activity, therefore, this parameter is precisely controlled according to the workload of a given muscle. Thin filament length is thought to be regulated by two major, but only partially understood mechanisms: it is set by (i) factors that mediate the assembly of filaments from monomers and catalyze their elongation, and (ii) by factors that specify their length and uniformity. Mutations affecting these factors can alter the length of thin filaments, and in human cases, many of them are linked to debilitating diseases such as nemaline myopathy and dilated cardiomyopathy.
Genetics, 2016
The noncanonical Frizzled/planar cell polarity (PCP) pathway regulates establishment of polarity ... more The noncanonical Frizzled/planar cell polarity (PCP) pathway regulates establishment of polarity within the plane of an epithelium to generate diversity of cell fates, asymmetric, but highly aligned structures, or to orchestrate the directional migration of cells during convergent extension during vertebrate gastrulation. In Drosophila, PCP signaling is essential to orient actin wing hairs and to align ommatidia in the eye, in part by coordinating the movement of groups of photoreceptor cells during ommatidial rotation. Importantly, the coordination of PCP signaling with changes in the cytoskeleton is essential for proper epithelial polarity. Formins polymerize linear actin filaments and are key regulators of the actin cytoskeleton. Here, we show that the diaphanous-related formin, Frl, the single fly member of the FMNL (formin related in leukocytes/formin-like) formin subfamily affects ommatidial rotation in the Drosophila eye and is controlled by the Rho family GTPase Cdc42. Inter...
bioRxiv, 2021
Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin... more Myofibrils are long intracellular cables specific to muscles, composed mainly of actin and myosin filaments. The actin and myosin filaments are organized into repeated units called sarcomeres, which form the myofibril cables. Muscle contraction is achieved by the simultaneous shortening of sarcomeres and for a highly coordinated contraction to occur all sarcomeres should have the same size. Muscles have evolved a variety of ways to ensure sarcomere homogeneity, one example being the controlled oligomerization of Zasp proteins that sets the diameter of the myofibril. To understand how Zasp proteins effect myofibril growth, we looked for Zasp-binding proteins at the Z-disc. We found that the E1 subunit of the oxoglutarate dehydrogenase complex is recruited to the Z-disc by Zasp52 and is required to sustain myofibril growth. By making specific mutants, we show that its enzymatic activity is important for myofibril growth, and that the other two subunits of the complex are also required...
Genetics, 2000
The Drosophila melanogaster Ketel gene was identified via the KetelD dominant female sterile muta... more The Drosophila melanogaster Ketel gene was identified via the KetelD dominant female sterile mutations and their ketelr revertant alleles that are recessive zygotic lethals. The maternally acting KetelD mutations inhibit cleavage nuclei formation. We cloned the Ketel gene on the basis of a common breakpoint in 38E1.2-3 in four ketelr alleles. The Ketel+ transgenes rescue ketelr-associated zygotic lethality and slightly reduce KetelD-associated dominant female sterility. Ketel is a single copy gene. It is transcribed to a single 3.6-kb mRNA, predicted to encode the 97-kD Ketel protein. The 884-amino-acid sequence of Ketel is 60% identical and 78% similar to that of human importin-β, the nuclear import receptor for proteins with a classical NLS. Indeed, Ketel supports import of appropriately designed substrates into nuclei of digitonin-permeabilized HeLa cells. As shown by a polyclonal anti-Ketel antibody, nurse cells synthesize and transfer Ketel protein into the oocyte cytoplasm fro...
International Journal of Molecular Sciences
The actin containing tropomyosin and troponin decorated thin filaments form one of the crucial co... more The actin containing tropomyosin and troponin decorated thin filaments form one of the crucial components of the contractile apparatus in muscles. The thin filaments are organized into densely packed lattices interdigitated with myosin-based thick filaments. The crossbridge interactions between these myofilaments drive muscle contraction, and the degree of myofilament overlap is a key factor of contractile force determination. As such, the optimal length of the thin filaments is critical for efficient activity, therefore, this parameter is precisely controlled according to the workload of a given muscle. Thin filament length is thought to be regulated by two major, but only partially understood mechanisms: it is set by (i) factors that mediate the assembly of filaments from monomers and catalyze their elongation, and (ii) by factors that specify their length and uniformity. Mutations affecting these factors can alter the length of thin filaments, and in human cases, many of them are...
Applied Microbiology and Biotechnology, 2005
Biochemical and pharmacological properties of biosurfactants produced at 45°C temperature by Pseu... more Biochemical and pharmacological properties of biosurfactants produced at 45°C temperature by Pseudomonas aeruginosa mucoid (M) and non-mucoid (NM) strains, isolated from hydrocarbon-contaminated soil samples, were characterized. Both the strains secreted appreciable amount of biosurfactants (5.0-6.5 g/l), responsible for the reduction of surface tension of the medium from 68 to 29±0.5 mN/m post 96 h of growth. Maximum yield of biosurfactants was observed following the supplementation of NH 4 Cl and glycerol as nitrogenous source and carbon source, respectively. These thermostable biosurfactants exhibited strong emulsifying property and could release appreciable amount of oil from saturated sand-pack column. Pharmacological characterization of these biosurfactants revealed that they induced dose-dependent hemolysis and coagulation of platelet-poor plasma but were non-detrimental to chicken lung, liver, heart and kidney tissues. Our study has documented that biosurfactants from P. aeruginosa M and NM strains could be exploited for use in petroleum sectors as well in pharmaceutical industries.
PLoS Genetics, 2014
During muscle development, myosin and actin containing filaments assemble into the highly organiz... more During muscle development, myosin and actin containing filaments assemble into the highly organized sarcomeric structure critical for muscle function. Although sarcomerogenesis clearly involves the de novo formation of actin filaments, this process remained poorly understood. Here we show that mouse and Drosophila members of the DAAM formin family are sarcomere-associated actin assembly factors enriched at the Z-disc and M-band. Analysis of dDAAM mutants revealed a pivotal role in myofibrillogenesis of larval somatic muscles, indirect flight muscles and the heart. We found that loss of dDAAM function results in multiple defects in sarcomere development including thin and thick filament disorganization, Zdisc and M-band formation, and a near complete absence of the myofibrillar lattice. Collectively, our data suggest that dDAAM is required for the initial assembly of thin filaments, and subsequently it promotes filament elongation by assembling short actin polymers that anneal to the pointed end of the growing filaments, and by antagonizing the capping protein Tropomodulin.