Adán Guerrero - Academia.edu (original) (raw)

Papers by Adán Guerrero

ABSTRACTDue to the wave nature of light, optical microscopy has a lower-bound lateral resolution ... more ABSTRACTDue to the wave nature of light, optical microscopy has a lower-bound lateral resolution limit of about half of the wavelength of the detected light, i.e., within the range of 200 to 300 nm. The Fluorescence Fluctuation based Super Resolution Microscopy (FF-SRM) encompases a collection of image analysis techniques which rely on the statistical processing of temporal variations of fluorescence to reduce the uncertainty about the fluorophore positions within a sample, hence, bringing spatial resolution down to several tens of nm. The FF-SRM is known to be suitable for live-cell imaging due to its compatibility with most fluorescent probes and lower instrumental and experimental requirements, which are mostly camera-based epifluorescence instruments. Each FF-SRM approach has strengths and weaknesses, which depend directly on the underlying statistical principles through which enhanced spatial resolution is achieved. In this review, the basic concepts and principles behind a ran...

PLOS Pathogens, 2021

The insecticidal Cry11Aa and Cyt1Aa proteins are produced byBacillus thuringiensisas crystal incl... more The insecticidal Cry11Aa and Cyt1Aa proteins are produced byBacillus thuringiensisas crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins forin vivolocalization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. A...

Current Research in Cell Biology, 2021

After colonization of the mosquito midgut by the malaria parasite, Plasmodium differentiates from... more After colonization of the mosquito midgut by the malaria parasite, Plasmodium differentiates from an invasive, motile ookinete to a multiplicative, sessile oocyst. Despite their importance in establishing the infection and increasing its population, relatively little is known about the early morphological transformation associated with these changes in function. Oocyst differentiation begins with the formation of a spherical protrusion near the center of the crescent-shaped ookinete. As this protuberance grows, it engulfs the content of the two distal ends, thus rounding the cell. In this work, scrutinized observations of the overall changes in shape, coupled with the migration of the malaria pigment granules and the nucleus into the protuberance, revealed that the movement of the cell content happens in an anteroposterior manner. The resulting data, formalized as morphometric measurements, led to the identification of 5 transitional stages and to the development of a computer training algorithm that automatically classifies them. Since cell differentiation has been associated with redox fluctuations, the classification algorithm was tested with parasites stained with a glutathione-specific fluorescent probe. This revealed changes in the glutathione content during differentiation that are suggestive of a redox modulation during transformation.

bioRxiv, 2020

After colonization of the mosquito midgut by the malaria parasite, Plasmodium differentiates from... more After colonization of the mosquito midgut by the malaria parasite, Plasmodium differentiates from an invasive, motile ookinete to a multiplicative, sessile oocyst. Despite their importance in establishing the infection and increasing its population, the morphological transformation associated with these changes in function has been scarcely explored. Oocyst differentiation begins with the formation of a spherical protrusion near the center of the crescent-shaped ookinete. As this protuberance grows, it engulfs the content of the two distal ends, thus rounding the cell. In this work, scrutinized observations of the overall changes in shape, coupled with the migration of the malaria pigment granules and the nucleus into the protuberance, revealed that the movement of the cell content happens in an anteroposterior manner. The resulting data, formalized as morphometric measurements, led to the identification of 5 transitional stages and to the development of a computer training algorith...

eLife, 2020

Spermatozoa of marine invertebrates are attracted to their conspecific female gamete by diffusive... more Spermatozoa of marine invertebrates are attracted to their conspecific female gamete by diffusive molecules, called chemoattractants, released from the egg investments in a process known as chemotaxis. The information from the egg chemoattractant concentration field is decoded into intracellular Ca2+ concentration ([Ca2+]i) changes that regulate the internal motors that shape the flagellum as it beats. By studying sea urchin species-specific differences in sperm chemoattractant-receptor characteristics we show that receptor density constrains the steepness of the chemoattractant concentration gradient detectable by spermatozoa. Through analyzing different chemoattractant gradient forms, we demonstrate for the first time that Strongylocentrotus purpuratus sperm are chemotactic and this response is consistent with frequency entrainment of two coupled physiological oscillators: i) the stimulus function and ii) the [Ca2+]i changes. We demonstrate that the slope of the chemoattractant gradients provides the coupling force between both oscillators, arising as a fundamental requirement for sperm chemotaxis.

Reproduction in Aquatic Animals, 2020

The sperm tail movement is a direct result from contributions of fluid mechanics, elasticity, and... more The sperm tail movement is a direct result from contributions of fluid mechanics, elasticity, and molecular-motor activity. Within the flagellum, the axonemal engine yields overall mechanical response and, ultimately, motility. This chapter attempts to provide a comprehensive and integrative overview of the relationship between the mechanics, signaling of sperm propulsion, and the physiological function of these cells in 3D. Sperm swimming, with its intricate coupling between the regulations of the flagellar beating has to ultimately fulfill its evolutionary function honed in their natural environment, the open sea. The strategies that are being employed to unravel this fascinating and fundamental process are revisited, where the sliding of water bodies shape chemical landscapes sensed by sperms during their journey, affecting motility patterns and directly determining gamete encounter rates.

Bioinformatics, 2020

Summary FCSlib is an open-source R tool for fluorescence fluctuation spectroscopy data analysis. ... more Summary FCSlib is an open-source R tool for fluorescence fluctuation spectroscopy data analysis. It encompasses techniques such as Fluorescence Correlation Spectroscopy, Number and Brightness, Pair Correlation Function and Pair Correlation of Molecular Brightness. Availability and implementation Source code available at https://cran.r-project.org/web/packages/FCSlib/ for Linux, Windows and macOS platforms. Sample data as well as a user's guide are available at https://github.com/FCSlib/FCSlib. Supplementary information Supplementary data are available at Bioinformatics online.

Autophagy is an evolutionary conserved pathway, by which eukaryotic cells degrade long-living cel... more Autophagy is an evolutionary conserved pathway, by which eukaryotic cells degrade long-living cellular proteins and intracellular organelles, to maintain a pool of available nutrients. Impaired autophagy has been associated to important pathophysiological conditions, and this is the reason why several techniques have been developed for its correct assessment and monitoring. Fluorescence microscopy is one of these tools, which relies on the detection of specific fluorescence changes of targeted GFP-based reporters in dot-like organelles in which autophagy is executed. Currently, several procedures exist to count and segment this punctate structures in the resulting fluorescence images, however, they are either based on subjective criteria, or no information is available related to them. Here we present the concept of an algorithm for a semi-automatic detection and segmentation in 2D fluorescence images of spot-like structures similar to those observed under induction of autophagy. By...

Communications in Computer and Information Science, 2016

Super-resolution microscopy techniques overcome the diffraction limit of optical microscopy. The ... more Super-resolution microscopy techniques overcome the diffraction limit of optical microscopy. The bayesian analysis of blinking and bleaching (3B analysis) is a super-resolution microscopy method that resolves biological structures with a lateral spatial resolution in the range of 50 nm. This method requires an extensive processing time to generate each super-resolution image. We present the parallelization of the 3B analysis for a personal computer and for a cluster which reduces the time for 3B analysis, and provide Parallel 3B an ImageJ plugin that extends the current implementation of the algorithm to parallel computing.

Cyclin-dependent kinases (CDKs). A family of Ser/Thr kinases that have major roles in the regulat... more Cyclin-dependent kinases (CDKs). A family of Ser/Thr kinases that have major roles in the regulation of the cell cycle. CDKs bind to their regulatory subunits, the cyclins, the concentrations of which oscillate throughout the cell cycle, which regulates CDK activity.

Intracellular calcium ([Ca2+]i) is a basic, versatile and ubiquitous cellular signal controlling ... more Intracellular calcium ([Ca2+]i) is a basic, versatile and ubiquitous cellular signal controlling a wide variety of biological processes. A remarkable example is the steering of sea urchin spermatozoa towards the conspecific egg by a spatially and temporally orchestrated series of cytosolic [Ca2+]ispikes. Although this process has been an experimental paradigm for reproduction and sperm chemotaxis studies, the composition and regulation of the signalling network underlying the cytosolic calcium fluctuations are hitherto not fully understood. Here, we used a differential equations model of the signalling network to assess which set of channels can explain the characteristic envelop and temporal organisation of the [Ca2+]i-spike trains. The signalling network comprises an initial membrane hyperpolarisation/repolarisation produced by an upstream module triggered by the egg-released chemoattractant peptide, via receptor activation, cGMP synthesis and decay. Followed by downstream modules...

Humans and other vertebrates define body axis left-right asymmetry in the early stages of embryo ... more Humans and other vertebrates define body axis left-right asymmetry in the early stages of embryo development (Shiratori and Hamada, 2006). The mechanism behind left-right establishment is not fully understood (Mizuno et al., 2020; Maerker et al., 2021; Minegishi et al., 2021). Symmetry breaking occurs in a dedicated organ called the left-right organizer (LRO) and involves motile cilia generating fluid-flow therein. However, it has been a matter of debate whether the process of symmetry breaking relies on a chemosensory or a mechanosensory mechanism (Shinohara et al., 2012). Novel tailored manipulations for LRO fluid extraction in living zebrafish embryos allowed us to pinpoint a decisive developmental period for breaking left-right symmetry during development. The shortest critical time-window was narrowed to one hour and characterized by a mild counterclockwise flow. The experimental challenge consisted in emptying the LRO of its fluid, abrogating simultaneously flow force and chem...

Developmental cell, Jan 23, 2014

Internal organs are asymmetrically positioned inside the body. Embryonic motile cilia play an ess... more Internal organs are asymmetrically positioned inside the body. Embryonic motile cilia play an essential role in this process by generating a directional fluid flow inside the vertebrate left-right organizer. Detailed characterization of how fluid flow dynamics modulates laterality is lacking. We used zebrafish genetics to experimentally generate a range of flow dynamics. By following the development of each embryo, we show that fluid flow in the left-right organizer is asymmetric and provides a good predictor of organ laterality. This was tested in mosaic organizers composed of motile and immotile cilia generated by dnah7 knockdowns. In parallel, we used simulations of fluid dynamics to analyze our experimental data. These revealed that fluid flow generated by 30 or more cilia predicts 90% situs solitus, similar to experimental observations. We conclude that cilia number, dorsal anterior motile cilia clustering, and left flow are critical to situs solitus via robust asymmetric charo...

Nature Communications

The resolution of fluorescence microscopy images is limited by the physical properties of light. ... more The resolution of fluorescence microscopy images is limited by the physical properties of light. In the last decade, numerous super-resolution microscopy (SRM) approaches have been proposed to deal with such hindrance. Here we present Mean-Shift Super Resolution (MSSR), a new SRM algorithm based on the Mean Shift theory, which extends spatial resolution of single fluorescence images beyond the diffraction limit of light. MSSR works on low and high fluorophore densities, is not limited by the architecture of the optical setup and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image stacks, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other SRM approaches. Along with its wide accessibility, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.

Biosystems, 2021

Intracellular Ca2+ is a key regulator of cell signaling and sperm are not the exception. Cells of... more Intracellular Ca2+ is a key regulator of cell signaling and sperm are not the exception. Cells often use cytoplasmic Ca2+ concentration ([Ca2+]i) oscillations as a means to decodify external and internal information. [Ca2+]i oscillations faster than those usually found in other cells and correlated with flagellar beat were the first to be described in sperm in 1993 by Susan Suarez, in the boar. More than 20 years passed before similar [Ca2+]i oscillations were documented in human sperm, simultaneously examining their flagellar beat in three dimensions by Corkidi et al.2017. On the other hand, 10 years after the discovery of the fast boar [Ca2+]i oscillations, slower ones triggered by compounds from the egg external envelope were found to regulate cell motility and chemotaxis in sperm from marine organisms. Today it is known that sperm display fast and slow spontaneous and agonist triggered [Ca2+]i oscillations. In mammalian sperm these Ca2+ transients may act like a multifaceted tool that regulates fundamental functions such as motility and acrosome reaction. This review covers the main sperm species and experimental conditions where [Ca2+]i oscillations have been described and discusses what is known about the transporters involved, their regulation and the physiological purpose of these oscillations. There is a lot to be learned regarding the origin, regulation and physiological relevance of these Ca2+ oscillations.

PLOS Computational Biology, 2020

Intracellular calcium ([Ca 2+ ] i) is a basic and ubiquitous cellular signal controlling a wide v... more Intracellular calcium ([Ca 2+ ] i) is a basic and ubiquitous cellular signal controlling a wide variety of biological processes. A remarkable example is the steering of sea urchin spermatozoa towards the conspecific egg by a spatially and temporally orchestrated series of [Ca 2+ ] i spikes. Although this process has been an experimental paradigm for reproduction and sperm chemotaxis studies, the composition and regulation of the signalling network underlying the cytosolic calcium fluctuations are hitherto not fully understood. Here, we used a differential equations model of the signalling network to assess which set of channels can explain the characteristic envelope and temporal organisation of the [Ca 2+ ] i-spike trains. The signalling network comprises an initial membrane hyperpolarisation produced by an Upstream module triggered by the egg-released chemoattractant peptide, via receptor activation, cGMP synthesis and decay. Followed by downstream modules leading to intraflagellar pH (pH i), voltage and [Ca 2+ ] i fluctuations. The Upstream module outputs were fitted to kinetic data on cGMP activity and early membrane potential changes measured in bulk cell populations. Two candidate modules featuring voltage-dependent Ca 2+-channels link these outputs to the downstream dynamics and can independently explain the typical decaying envelope and the progressive spacing of the spikes. In the first module, [Ca 2+ ] i-spike trains require the concerted action of a classical Ca V-like channel and a potassium channel, BK (Slo1), whereas the second module relies on pH i-dependent CatSper dynamics articulated with voltage-dependent neutral sodium-proton exchanger (NHE). We analysed the dynamics of these two modules alone and in mixed scenarios. We show that the [Ca 2+ ] i dynamics observed experimentally after sustained alkalinisation can be reproduced by a model featuring the Cat-Sper and NHE module but not by those including the pH-independent Ca V and BK module or proportionate mixed scenarios. We conclude in favour of the module containing CatSper and NHE and highlight experimentally testable predictions that would corroborate this conclusion.

Mathematics, 2019

The emergence of left–right (LR) asymmetry in vertebrates is a prime example of a highly conserve... more The emergence of left–right (LR) asymmetry in vertebrates is a prime example of a highly conserved fundamental process in developmental biology. Details of how symmetry breaking is established in different organisms are, however, still not fully understood. In the zebrafish (Danio rerio), it is known that a cilia-mediated vortical flow exists within its LR organizer, the so-called Kupffer’s vesicle (KV), and that it is directly involved in early LR determination. However, the flow exhibits spatio-temporal complexity; moreover, its conversion to asymmetric development has proved difficult to resolve despite a number of recent experimental advances and numerical efforts. In this paper, we provide further theoretical insight into the essence of flow generation by putting together a minimal biophysical model which reduces to a set of singular solutions satisfying the imposed boundary conditions; one that is informed by our current understanding of the fluid flow in the KV, that satisfie...

ABSTRACTDue to the wave nature of light, optical microscopy has a lower-bound lateral resolution ... more ABSTRACTDue to the wave nature of light, optical microscopy has a lower-bound lateral resolution limit of about half of the wavelength of the detected light, i.e., within the range of 200 to 300 nm. The Fluorescence Fluctuation based Super Resolution Microscopy (FF-SRM) encompases a collection of image analysis techniques which rely on the statistical processing of temporal variations of fluorescence to reduce the uncertainty about the fluorophore positions within a sample, hence, bringing spatial resolution down to several tens of nm. The FF-SRM is known to be suitable for live-cell imaging due to its compatibility with most fluorescent probes and lower instrumental and experimental requirements, which are mostly camera-based epifluorescence instruments. Each FF-SRM approach has strengths and weaknesses, which depend directly on the underlying statistical principles through which enhanced spatial resolution is achieved. In this review, the basic concepts and principles behind a ran...

PLOS Pathogens, 2021

The insecticidal Cry11Aa and Cyt1Aa proteins are produced byBacillus thuringiensisas crystal incl... more The insecticidal Cry11Aa and Cyt1Aa proteins are produced byBacillus thuringiensisas crystal inclusions. They work synergistically inducing high toxicity against mosquito larvae. It was proposed that these crystal inclusions are rapidly solubilized and activated in the gut lumen, followed by pore formation in midgut cells killing the larvae. In addition, Cyt1Aa functions as a Cry11Aa binding receptor, inducing Cry11Aa oligomerization and membrane insertion. Here, we used fluorescent labeled crystals, protoxins or activated toxins forin vivolocalization at nano-scale resolution. We show that after larvae were fed solubilized proteins, these proteins were not accumulated inside the gut and larvae were not killed. In contrast, if larvae were fed soluble non-toxic mutant proteins, these proteins were found inside the gut bound to gut-microvilli. Only feeding with crystal inclusions resulted in high larval mortality, suggesting that they have a role for an optimal intoxication process. A...

Current Research in Cell Biology, 2021

After colonization of the mosquito midgut by the malaria parasite, Plasmodium differentiates from... more After colonization of the mosquito midgut by the malaria parasite, Plasmodium differentiates from an invasive, motile ookinete to a multiplicative, sessile oocyst. Despite their importance in establishing the infection and increasing its population, relatively little is known about the early morphological transformation associated with these changes in function. Oocyst differentiation begins with the formation of a spherical protrusion near the center of the crescent-shaped ookinete. As this protuberance grows, it engulfs the content of the two distal ends, thus rounding the cell. In this work, scrutinized observations of the overall changes in shape, coupled with the migration of the malaria pigment granules and the nucleus into the protuberance, revealed that the movement of the cell content happens in an anteroposterior manner. The resulting data, formalized as morphometric measurements, led to the identification of 5 transitional stages and to the development of a computer training algorithm that automatically classifies them. Since cell differentiation has been associated with redox fluctuations, the classification algorithm was tested with parasites stained with a glutathione-specific fluorescent probe. This revealed changes in the glutathione content during differentiation that are suggestive of a redox modulation during transformation.

bioRxiv, 2020

After colonization of the mosquito midgut by the malaria parasite, Plasmodium differentiates from... more After colonization of the mosquito midgut by the malaria parasite, Plasmodium differentiates from an invasive, motile ookinete to a multiplicative, sessile oocyst. Despite their importance in establishing the infection and increasing its population, the morphological transformation associated with these changes in function has been scarcely explored. Oocyst differentiation begins with the formation of a spherical protrusion near the center of the crescent-shaped ookinete. As this protuberance grows, it engulfs the content of the two distal ends, thus rounding the cell. In this work, scrutinized observations of the overall changes in shape, coupled with the migration of the malaria pigment granules and the nucleus into the protuberance, revealed that the movement of the cell content happens in an anteroposterior manner. The resulting data, formalized as morphometric measurements, led to the identification of 5 transitional stages and to the development of a computer training algorith...

eLife, 2020

Spermatozoa of marine invertebrates are attracted to their conspecific female gamete by diffusive... more Spermatozoa of marine invertebrates are attracted to their conspecific female gamete by diffusive molecules, called chemoattractants, released from the egg investments in a process known as chemotaxis. The information from the egg chemoattractant concentration field is decoded into intracellular Ca2+ concentration ([Ca2+]i) changes that regulate the internal motors that shape the flagellum as it beats. By studying sea urchin species-specific differences in sperm chemoattractant-receptor characteristics we show that receptor density constrains the steepness of the chemoattractant concentration gradient detectable by spermatozoa. Through analyzing different chemoattractant gradient forms, we demonstrate for the first time that Strongylocentrotus purpuratus sperm are chemotactic and this response is consistent with frequency entrainment of two coupled physiological oscillators: i) the stimulus function and ii) the [Ca2+]i changes. We demonstrate that the slope of the chemoattractant gradients provides the coupling force between both oscillators, arising as a fundamental requirement for sperm chemotaxis.

Reproduction in Aquatic Animals, 2020

The sperm tail movement is a direct result from contributions of fluid mechanics, elasticity, and... more The sperm tail movement is a direct result from contributions of fluid mechanics, elasticity, and molecular-motor activity. Within the flagellum, the axonemal engine yields overall mechanical response and, ultimately, motility. This chapter attempts to provide a comprehensive and integrative overview of the relationship between the mechanics, signaling of sperm propulsion, and the physiological function of these cells in 3D. Sperm swimming, with its intricate coupling between the regulations of the flagellar beating has to ultimately fulfill its evolutionary function honed in their natural environment, the open sea. The strategies that are being employed to unravel this fascinating and fundamental process are revisited, where the sliding of water bodies shape chemical landscapes sensed by sperms during their journey, affecting motility patterns and directly determining gamete encounter rates.

Bioinformatics, 2020

Summary FCSlib is an open-source R tool for fluorescence fluctuation spectroscopy data analysis. ... more Summary FCSlib is an open-source R tool for fluorescence fluctuation spectroscopy data analysis. It encompasses techniques such as Fluorescence Correlation Spectroscopy, Number and Brightness, Pair Correlation Function and Pair Correlation of Molecular Brightness. Availability and implementation Source code available at https://cran.r-project.org/web/packages/FCSlib/ for Linux, Windows and macOS platforms. Sample data as well as a user's guide are available at https://github.com/FCSlib/FCSlib. Supplementary information Supplementary data are available at Bioinformatics online.

Autophagy is an evolutionary conserved pathway, by which eukaryotic cells degrade long-living cel... more Autophagy is an evolutionary conserved pathway, by which eukaryotic cells degrade long-living cellular proteins and intracellular organelles, to maintain a pool of available nutrients. Impaired autophagy has been associated to important pathophysiological conditions, and this is the reason why several techniques have been developed for its correct assessment and monitoring. Fluorescence microscopy is one of these tools, which relies on the detection of specific fluorescence changes of targeted GFP-based reporters in dot-like organelles in which autophagy is executed. Currently, several procedures exist to count and segment this punctate structures in the resulting fluorescence images, however, they are either based on subjective criteria, or no information is available related to them. Here we present the concept of an algorithm for a semi-automatic detection and segmentation in 2D fluorescence images of spot-like structures similar to those observed under induction of autophagy. By...

Communications in Computer and Information Science, 2016

Super-resolution microscopy techniques overcome the diffraction limit of optical microscopy. The ... more Super-resolution microscopy techniques overcome the diffraction limit of optical microscopy. The bayesian analysis of blinking and bleaching (3B analysis) is a super-resolution microscopy method that resolves biological structures with a lateral spatial resolution in the range of 50 nm. This method requires an extensive processing time to generate each super-resolution image. We present the parallelization of the 3B analysis for a personal computer and for a cluster which reduces the time for 3B analysis, and provide Parallel 3B an ImageJ plugin that extends the current implementation of the algorithm to parallel computing.

Cyclin-dependent kinases (CDKs). A family of Ser/Thr kinases that have major roles in the regulat... more Cyclin-dependent kinases (CDKs). A family of Ser/Thr kinases that have major roles in the regulation of the cell cycle. CDKs bind to their regulatory subunits, the cyclins, the concentrations of which oscillate throughout the cell cycle, which regulates CDK activity.

Intracellular calcium ([Ca2+]i) is a basic, versatile and ubiquitous cellular signal controlling ... more Intracellular calcium ([Ca2+]i) is a basic, versatile and ubiquitous cellular signal controlling a wide variety of biological processes. A remarkable example is the steering of sea urchin spermatozoa towards the conspecific egg by a spatially and temporally orchestrated series of cytosolic [Ca2+]ispikes. Although this process has been an experimental paradigm for reproduction and sperm chemotaxis studies, the composition and regulation of the signalling network underlying the cytosolic calcium fluctuations are hitherto not fully understood. Here, we used a differential equations model of the signalling network to assess which set of channels can explain the characteristic envelop and temporal organisation of the [Ca2+]i-spike trains. The signalling network comprises an initial membrane hyperpolarisation/repolarisation produced by an upstream module triggered by the egg-released chemoattractant peptide, via receptor activation, cGMP synthesis and decay. Followed by downstream modules...

Humans and other vertebrates define body axis left-right asymmetry in the early stages of embryo ... more Humans and other vertebrates define body axis left-right asymmetry in the early stages of embryo development (Shiratori and Hamada, 2006). The mechanism behind left-right establishment is not fully understood (Mizuno et al., 2020; Maerker et al., 2021; Minegishi et al., 2021). Symmetry breaking occurs in a dedicated organ called the left-right organizer (LRO) and involves motile cilia generating fluid-flow therein. However, it has been a matter of debate whether the process of symmetry breaking relies on a chemosensory or a mechanosensory mechanism (Shinohara et al., 2012). Novel tailored manipulations for LRO fluid extraction in living zebrafish embryos allowed us to pinpoint a decisive developmental period for breaking left-right symmetry during development. The shortest critical time-window was narrowed to one hour and characterized by a mild counterclockwise flow. The experimental challenge consisted in emptying the LRO of its fluid, abrogating simultaneously flow force and chem...

Developmental cell, Jan 23, 2014

Internal organs are asymmetrically positioned inside the body. Embryonic motile cilia play an ess... more Internal organs are asymmetrically positioned inside the body. Embryonic motile cilia play an essential role in this process by generating a directional fluid flow inside the vertebrate left-right organizer. Detailed characterization of how fluid flow dynamics modulates laterality is lacking. We used zebrafish genetics to experimentally generate a range of flow dynamics. By following the development of each embryo, we show that fluid flow in the left-right organizer is asymmetric and provides a good predictor of organ laterality. This was tested in mosaic organizers composed of motile and immotile cilia generated by dnah7 knockdowns. In parallel, we used simulations of fluid dynamics to analyze our experimental data. These revealed that fluid flow generated by 30 or more cilia predicts 90% situs solitus, similar to experimental observations. We conclude that cilia number, dorsal anterior motile cilia clustering, and left flow are critical to situs solitus via robust asymmetric charo...

Nature Communications

The resolution of fluorescence microscopy images is limited by the physical properties of light. ... more The resolution of fluorescence microscopy images is limited by the physical properties of light. In the last decade, numerous super-resolution microscopy (SRM) approaches have been proposed to deal with such hindrance. Here we present Mean-Shift Super Resolution (MSSR), a new SRM algorithm based on the Mean Shift theory, which extends spatial resolution of single fluorescence images beyond the diffraction limit of light. MSSR works on low and high fluorophore densities, is not limited by the architecture of the optical setup and is applicable to single images as well as temporal series. The theoretical limit of spatial resolution, based on optimized real-world imaging conditions and analysis of temporal image stacks, has been measured to be 40 nm. Furthermore, MSSR has denoising capabilities that outperform other SRM approaches. Along with its wide accessibility, MSSR is a powerful, flexible, and generic tool for multidimensional and live cell imaging applications.

Biosystems, 2021

Intracellular Ca2+ is a key regulator of cell signaling and sperm are not the exception. Cells of... more Intracellular Ca2+ is a key regulator of cell signaling and sperm are not the exception. Cells often use cytoplasmic Ca2+ concentration ([Ca2+]i) oscillations as a means to decodify external and internal information. [Ca2+]i oscillations faster than those usually found in other cells and correlated with flagellar beat were the first to be described in sperm in 1993 by Susan Suarez, in the boar. More than 20 years passed before similar [Ca2+]i oscillations were documented in human sperm, simultaneously examining their flagellar beat in three dimensions by Corkidi et al.2017. On the other hand, 10 years after the discovery of the fast boar [Ca2+]i oscillations, slower ones triggered by compounds from the egg external envelope were found to regulate cell motility and chemotaxis in sperm from marine organisms. Today it is known that sperm display fast and slow spontaneous and agonist triggered [Ca2+]i oscillations. In mammalian sperm these Ca2+ transients may act like a multifaceted tool that regulates fundamental functions such as motility and acrosome reaction. This review covers the main sperm species and experimental conditions where [Ca2+]i oscillations have been described and discusses what is known about the transporters involved, their regulation and the physiological purpose of these oscillations. There is a lot to be learned regarding the origin, regulation and physiological relevance of these Ca2+ oscillations.

PLOS Computational Biology, 2020

Intracellular calcium ([Ca 2+ ] i) is a basic and ubiquitous cellular signal controlling a wide v... more Intracellular calcium ([Ca 2+ ] i) is a basic and ubiquitous cellular signal controlling a wide variety of biological processes. A remarkable example is the steering of sea urchin spermatozoa towards the conspecific egg by a spatially and temporally orchestrated series of [Ca 2+ ] i spikes. Although this process has been an experimental paradigm for reproduction and sperm chemotaxis studies, the composition and regulation of the signalling network underlying the cytosolic calcium fluctuations are hitherto not fully understood. Here, we used a differential equations model of the signalling network to assess which set of channels can explain the characteristic envelope and temporal organisation of the [Ca 2+ ] i-spike trains. The signalling network comprises an initial membrane hyperpolarisation produced by an Upstream module triggered by the egg-released chemoattractant peptide, via receptor activation, cGMP synthesis and decay. Followed by downstream modules leading to intraflagellar pH (pH i), voltage and [Ca 2+ ] i fluctuations. The Upstream module outputs were fitted to kinetic data on cGMP activity and early membrane potential changes measured in bulk cell populations. Two candidate modules featuring voltage-dependent Ca 2+-channels link these outputs to the downstream dynamics and can independently explain the typical decaying envelope and the progressive spacing of the spikes. In the first module, [Ca 2+ ] i-spike trains require the concerted action of a classical Ca V-like channel and a potassium channel, BK (Slo1), whereas the second module relies on pH i-dependent CatSper dynamics articulated with voltage-dependent neutral sodium-proton exchanger (NHE). We analysed the dynamics of these two modules alone and in mixed scenarios. We show that the [Ca 2+ ] i dynamics observed experimentally after sustained alkalinisation can be reproduced by a model featuring the Cat-Sper and NHE module but not by those including the pH-independent Ca V and BK module or proportionate mixed scenarios. We conclude in favour of the module containing CatSper and NHE and highlight experimentally testable predictions that would corroborate this conclusion.

Mathematics, 2019

The emergence of left–right (LR) asymmetry in vertebrates is a prime example of a highly conserve... more The emergence of left–right (LR) asymmetry in vertebrates is a prime example of a highly conserved fundamental process in developmental biology. Details of how symmetry breaking is established in different organisms are, however, still not fully understood. In the zebrafish (Danio rerio), it is known that a cilia-mediated vortical flow exists within its LR organizer, the so-called Kupffer’s vesicle (KV), and that it is directly involved in early LR determination. However, the flow exhibits spatio-temporal complexity; moreover, its conversion to asymmetric development has proved difficult to resolve despite a number of recent experimental advances and numerical efforts. In this paper, we provide further theoretical insight into the essence of flow generation by putting together a minimal biophysical model which reduces to a set of singular solutions satisfying the imposed boundary conditions; one that is informed by our current understanding of the fluid flow in the KV, that satisfie...