Polo-like kinases: structural variations lead to multiple functions.

Members of the polo-like kinase (PLK) family are crucial regulators of cell cycle progression, centriole duplication, mitosis, cytokinesis and the DNA damage response. PLKs undergo major changes in abundance, activity, localization and structure at different stages of the cell cycle. They interact with other proteins in a tightly controlled spatiotemporal manner as part of a network that coordinates key cell cycle events. Their essential roles are highlighted by the fact that alterations in PLK function are associated with cancers and other diseases. Recent knowledge gained from PLK crystal structures, evolution and interacting molecules offers important insights into the mechanisms that underlie their regulation and activity, and suggests novel functions unrelated to cell cycle control for this family of kinases.

Nucleophagy contributes to genome stability through degradation of type II topoisomerases A and B and nucleolar components.

The nuclear architecture of mammalian cells can be altered as a consequence of anomalous accumulation of nuclear proteins or genomic alterations. Most of the knowledge about nuclear dynamics comes from studies on cancerous cells. How normal healthy cells maintain genome stability, avoiding accumulation of nuclear damaged material, is less understood. Here, we describe that primary mouse embryonic fibroblasts develop a basal level of nuclear buds and micronuclei, which increase after etoposide-induced DNA double-stranded breaks. Both basal and induced nuclear buds and micronuclei colocalize with the autophagic proteins BECN1 and LC3B (also known as MAP1LC3B) and with acidic vesicles, suggesting their clearance by nucleophagy. Some of the nuclear alterations also contain autophagic proteins and type II DNA topoisomerases (TOP2A and TOP2B), or the nucleolar protein fibrillarin, implying they are also targets of nucleophagy. We propose that basal nucleophagy contributes to genome and nuclear stability, as well as in response to DNA damage.

Recognising the importance and impact of Imaging Scientists: Global guidelines for establishing career paths within core facilities.

In the dynamic landscape of scientific research, imaging core facilities are vital hubs propelling collaboration and innovation at the technology development and dissemination frontier. Here, we present a collaborative effort led by Global BioImaging (GBI), introducing international recommendations geared towards elevating the careers of Imaging Scientists in core facilities. Despite the critical role of Imaging Scientists in modern research ecosystems, challenges persist in recognising their value, aligning performance metrics and providing avenues for career progression and job security. The challenges encompass a mismatch between classic academic career paths and service-oriented roles, resulting in a lack of understanding regarding the value and impact of Imaging Scientists and core facilities and how to evaluate them properly. They further include challenges around sustainability, dedicated training opportunities and the recruitment and retention of talent. Structured across these interrelated sections, the recommendations within this publication aim to propose globally applicable solutions to navigate these challenges. These recommendations apply equally to colleagues working in other core facilities and research institutions through which access to technologies is facilitated and supported. This publication emphasises the pivotal role of Imaging Scientists in advancing research programs and presents a blueprint for fostering their career progression within institutions all around the world.

In vivo nanoscale analysis of the dynamic synergistic interaction of Bacillus thuringiensis Cry11Aa and Cyt1Aa toxins in Aedes aegypti.

The insecticidal Cry11Aa and Cyt1Aa proteins are produced by Bacillus thuringiensis as 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 for in vivo localization 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. At the macroscopic level, Cry11Aa completely degraded the gastric caeca structure and, in the presence of Cyt1Aa, this effect was observed at lower toxin-concentrations and at shorter periods. The labeled Cry11Aa crystal protein, after midgut processing, binds to the gastric caeca and posterior midgut regions, and also to anterior and medium regions where it is internalized in ordered "net like" structures, leading finally to cell break down. During synergism both Cry11Aa and Cyt1Aa toxins showed a dynamic layered array at the surface of apical microvilli, where Cry11Aa is localized in the lower layer closer to the cell cytoplasm, and Cyt1Aa is layered over Cry11Aa. This array depends on the pore formation activity of Cry11Aa, since the non-toxic mutant Cry11Aa-E97A, which is unable to oligomerize, inverted this array. Internalization of Cry11Aa was also observed during synergism. These data indicate that the mechanism of action of Cry11Aa is more complex than previously anticipated, and may involve additional steps besides pore-formation activity.

Fluorescence fluctuation-based super-resolution microscopy: Basic concepts for an easy start.

Due to the wave nature of light, optical microscopy has a lower-bound lateral resolution limit of approximately half of the wavelength of visible light, that is, within the range of 200 to 350 nm. Fluorescence fluctuation-based super-resolution microscopy (FF-SRM) is a term used to encompass a collection of image analysis techniques that rely on the statistical processing of temporal variations of the fluorescence signal. FF-SRM aims to reduce the uncertainty of the location of fluorophores within an image, often improving spatial resolution by several tens of nanometers. FF-SRM is suitable for live-cell imaging due to its compatibility with most fluorescent probes and relatively simple 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 range of FF-SRM methods published to date are described. Their operational parameters are explained and guidance for their selection is provided.

Due to light's wave nature, an optical microscope's resolution range is 200 to 350 nanometers. Several techniques enhance resolution; this work encompasses several fluorescence fluctuation super-resolution (FF-SMR) methods capable of achieving nanoscopic scales. FF-SRM is known to be suitable for fixed or live-cell imaging and compatible with most conventional microscope setups found in a laboratory. However, each FF-SRM approach has its strengths and weaknesses, which depend directly on the underlying principles through which enhanced spatial resolution is achieved. Therefore, the basic concepts and principles behind diverse FF-SRM methods are revisited in this review. In addition, their operational parameters are explained, and guidance for their selection is provided for microscopists interested in FF-SRM.

Modular analysis of the control of flagellar Ca2+-spike trains produced by CatSper and CaV channels in sea urchin sperm.

Intracellular calcium ([Ca2+]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 [Ca2+]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 [Ca2+]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 (pHi), voltage and [Ca2+]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 Ca2+-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, [Ca2+]i-spike trains require the concerted action of a classical CaV-like channel and a potassium channel, BK (Slo1), whereas the second module relies on pHi-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 [Ca2+]i dynamics observed experimentally after sustained alkalinisation can be reproduced by a model featuring the CatSper and NHE module but not by those including the pH-independent CaV 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.

Super-resolution imaging of live sperm reveals dynamic changes of the actin cytoskeleton during acrosomal exocytosis.

Filamentous actin (F-actin) is a key factor in exocytosis in many cell types. In mammalian sperm, acrosomal exocytosis (denoted the acrosome reaction or AR), a special type of controlled secretion, is regulated by multiple signaling pathways and the actin cytoskeleton. However, the dynamic changes of the actin cytoskeleton in live sperm are largely not understood. Here, we used the powerful properties of SiR-actin to examine actin dynamics in live mouse sperm at the onset of the AR. By using a combination of super-resolution microscopy techniques to image sperm loaded with SiR-actin or sperm from transgenic mice containing Lifeact-EGFP, six regions containing F-actin within the sperm head were revealed. The proportion of sperm possessing these structures changed upon capacitation. By performing live-cell imaging experiments, we report that dynamic changes of F-actin during the AR occur in specific regions of the sperm head. While certain F-actin regions undergo depolymerization prior to the initiation of the AR, others remain unaltered or are lost after exocytosis occurs. Our work emphasizes the utility of live-cell nanoscopy, which will undoubtedly impact the search for mechanisms that underlie basic sperm functions.This article has an associated First Person interview with the first author of the paper.

Morphological, Biochemical, and Functional Study of Viral Replication Compartments Isolated from Adenovirus-Infected Cells.

UNLABELLED: Adenovirus (Ad) replication compartments (RC) are nuclear microenvironments where the viral genome is replicated and a coordinated program of late gene expression is established. These virus-induced nuclear sites seem to behave as central hubs for the regulation of virus-host cell interactions, since proteins that promote efficient viral replication as well as factors that participate in the antiviral response are coopted and concentrated there. To gain further insight into the activities of viral RC, here we report, for the first time, the morphology, composition, and activities of RC isolated from Ad-infected cells. Morphological analyses of isolated RC particles by superresolution microscopy showed that they were indistinguishable from RC within infected cells and that they displayed a dynamic compartmentalization. Furthermore, the RC-containing fractions (RCf) proved to be functional, as they directed de novo synthesis of viral DNA and RNA as well as RNA splicing, activities that are associated with RC in vivo. A detailed analysis of the production of viral late mRNA from RCf at different times postinfection revealed that viral mRNA splicing occurs in RC and that the synthesis, posttranscriptional processing, and release from RC to the nucleoplasm of individual viral late transcripts are spatiotemporally separate events. The results presented here demonstrate that RCf are a powerful system for detailed study into RC structure, composition, and activities and, as a result, the determination of the molecular mechanisms that induce the formation of these viral sites of adenoviruses and other nuclear-replicating viruses. IMPORTANCE: RC may represent molecular hubs where many aspects of virus-host cell interaction are controlled. Here, we show by superresolution microscopy that RCf have morphologies similar to those of RC within Ad-infected cells and that they appear to be compartmentalized, as nucleolin and DBP display different localization in the periphery of these viral sites. RCf proved to be functional, as they direct de novo synthesis of viral DNA and mRNA, allowing the detailed study of the regulation of viral genome replication and expression. Furthermore, we show that the synthesis and splicing of individual viral late mRNA occurs in RC and that they are subject to different temporal patterns of regulation, from their synthesis to their splicing and release from RC to the nucleoplasm. Hence, RCf represent a novel system to study molecular mechanisms that are orchestrated in viral RC to take control of the infected cell and promote an efficient viral replication cycle.

Niflumic acid disrupts marine spermatozoan chemotaxis without impairing the spatiotemporal detection of chemoattractant gradients.

In many broadcast-spawning marine organisms, oocytes release chemicals that guide conspecific spermatozoa towards them through chemotaxis. In the sea urchin Lytechinus pictus, the chemoattractant peptide speract triggers a train of fluctuations of intracellular Ca(2+) concentration in the sperm flagella. Each transient Ca(2+) elevation leads to a momentary increase in flagellar bending asymmetry, known as a chemotactic turn. Furthermore, chemotaxis requires a precise spatiotemporal coordination between the Ca(2+)-dependent turns and the form of chemoattractant gradient. Spermatozoa that perform Ca(2+)-dependent turns while swimming down the chemoattractant gradient, and conversely suppress turning events while swimming up the gradient, successfully approach the center of the gradient. Previous experiments in Strongylocentrotus purpuratus sea urchin spermatozoa showed that niflumic acid (NFA), an inhibitor of several ion channels, drastically altered the speract-induced Ca(2+) fluctuations and swimming patterns. In this study, mathematical modeling of the speract-dependent Ca(2+) signaling pathway suggests that NFA, by potentially affecting hyperpolarization-activated and cyclic nucleotide-gated channels, Ca(2+)-regulated Cl(-) channels and/or Ca(2+)-regulated K(+) channels, may alter the temporal organization of Ca(2+) fluctuations, and therefore disrupt chemotaxis. We used a novel automated method for analyzing sperm behavior and we identified that NFA does indeed disrupt chemotactic responses of L. pictus spermatozoa, although the temporal coordination between the Ca(2+)-dependent turns and the form of chemoattractant gradient is unaltered. Instead, NFA disrupts sperm chemotaxis by altering the arc length traveled during each chemotactic turning event. This alteration in the chemotactic turn trajectory disorientates spermatozoa at the termination of the turning event. We conclude that NFA disrupts chemotaxis without affecting how the spermatozoa decode environmental cues.

Fluid extraction from the left-right organizer uncovers mechanical properties needed for symmetry breaking.

Humans and other vertebrates define body axis left-right asymmetry in the early stages of embryo development. The mechanism behind left-right establishment is not fully understood. 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 physiological 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 chemical determinants. Our findings revealed an unprecedented recovery capacity of the embryo to re-fil and re-circulate new LRO fluid. The embryos that later developed laterality problems were found to be those that had lower anterior angular velocity and thus less anterior-posterior heterogeneity. Next, aiming to test the presence of any secreted determinant, we replaced the extracted LRO fluid by a physiological buffer. Despite some transitory flow homogenization, laterality defects were absent unless viscosity was altered, demonstrating that symmetry breaking does not depend on the nature of the fluid content but is rather sensitive to fluid mechanics. Altogether, we conclude that the zebrafish LRO is more sensitive to fluid dynamics for symmetry breaking.

Reorganization of the Flagellum Scaffolding Induces a Sperm Standstill During Fertilization.

Mammalian sperm delve into the female reproductive tract to fertilize the female gamete. The available information about how sperm regulate their motility during the final journey to the fertilization site is extremely limited. In this work, we investigated the structural and functional changes in the sperm flagellum after acrosomal exocytosis and during the interaction with the eggs. The evidence demonstrates that the double helix actin network surrounding the mitochondrial sheath of the midpiece undergoes structural changes prior to the motility cessation. This structural modification is accompanied by a decrease in diameter of the midpiece and is driven by intracellular calcium changes that occur concomitant with a reorganization of the actin helicoidal cortex. Although midpiece contraction may occur in a subset of cells that undergo acrosomal exocytosis, live-cell imaging during in vitro fertilization showed that the midpiece contraction is required for motility cessation after fusion is initiated. These findings provide the first evidence of the F-actin network's role in regulating sperm motility, adapting its function to meet specific cellular requirements during fertilization, and highlighting the broader significance of understanding sperm motility. Significant statement: In this work, we demonstrate that the helical structure of polymerized actin in the flagellum undergoes a rearrangement at the time of sperm-egg fusion. This process is driven by intracellular calcium and promotes a decrease in the sperm midpiece diameter as well as the arrest in motility, which is observed after the fusion process is initiated.

Semi-IPN hydrogels of collagen and gum arabic with antibacterial capacity and controlled release of drugs for potential application in wound healing.

The preparation of hydrogels based on biopolymers like collagen and gum arabic gives a chance to provide novel options that can be used in biomedical field. Through a polymeric semi-interpenetration technique, collagen-based polymeric matrices can be associated with gum arabic while controlling its physicochemical and biological properties. To create novel hydrogels with their potential use in the treatment of wounds, the semi-interpenetration process, altering the concentration (0-40% by wt) of gum arabic in a collagen matrix is explored. The ability of gum arabic to create intermolecular hydrogen bonds in the collagen matrix enables the development of semi-interpenetrating polymeric networks (semi-IPN)-based hydrogels with a faster gelation time and higher crosslinking. Amorphous granular surfaces with linked porosity are present in matrices with 30% (by wt) of gum arabic, enhancing the storage modulus and thermal degradation resistance. The hydrogels swell to very high extent in hydrolytic and proteolytic environments, good hemocompatibility, and suppression of growth of pathogens like E. coli, and all it is enhanced by gum arabic included them, in addition to enabling the controlled release of ketorolac. The chemical composition of theses semi-IPN matrices have no deleterious effects on monocytes or fibroblasts, promoting their proliferation, and lowering alpha tumor necrosis factor (α-TNF) secretion in human monocytes.

Smart collagen/xanthan gum-based hydrogels with antibacterial effect, drug release capacity and excellent performancein vitrobioactivity for wound healing application.

The design of hydrogels based on natural polymers that have modulation of antibacterial capacity, ideal performance in release capacity of encapsulated drugs, and desired bioactivity for applications in wound healing represents a modern trend in biomaterials. In this work, novel hydrogels of semi-interpenetrating polymeric networks based on collagen and xanthan gum (XG) were investigated. The linear chains of XG can semi-interpenetrate inside to matrix of crosslinked collagen with polyurethane under physiological conditions, generating amorphous surfaces with fibrillar-granular reliefs that have accelerated gelation time (about 15 min), super water absorption (up to 3100%) and high inhibition capacity of pathogenic bacteria such asEscherichia coli(up to 100% compared to amoxicillin at 20 ppm). The increment of XG in the hydrogel (up to 20 wt.%) allows for improvement in the storage module, resistance to thermal degradation, slow the rate of hydrolytic and proteolytic degradation, allowing to encapsulate and controlled release of molecules such as ketorolac and methylene blue; besides, it shows to keep the metabolic activity of fibroblasts and monocytes at 48 h of evaluation, without observing cytotoxic effects. The bioactivity of these hydrogels is improved since they have excellent hemocompatibility and enhanced cell proliferation. Specifically, the hydrogel with 20 wt.% of XG shows to decrease the production of tumor necrosis factor-αand CCL-2 cytokines, increasing the production of transforming growth factor-ßin human monocytes, which could be used to modulate inflammation and regenerative capacity in wound healing strategies.

Acute slices of mice testis seminiferous tubules unveil spontaneous and synchronous Ca2+ oscillations in germ cell clusters.

Spermatogenic cell differentiation involves changes in the concentration of cytoplasmic Ca(2+) ([Ca(2+)]i); however, very few studies exist on [Ca(2+)]i dynamics in these cells. Other tissues display Ca(2+) oscillations involving multicellular functional arrangements. These phenomena have been studied in acute slice preparations that preserve tissue architecture and intercellular communications. Here we report the implementation of intracellular Ca(2+) imaging in a sliced seminiferous tubule (SST) preparation to visualize [Ca(2+)]i changes of living germ cells in situ within the SST preparation. Ca(2+) imaging revealed that a subpopulation of male germ cells display spontaneous [Ca(2+)]i fluctuations resulting from Ca(2+) entry possibly throughout Ca(V)3 channels. These [Ca(2+)]i fluctuation patterns are also present in single acutely dissociated germ cells, but they differ from those recorded from germ cells in the SST preparation. Often, spontaneous Ca(2+) fluctuations of spermatogenic cells in the SST occur synchronously, so that clusters of cells can display Ca(2+) oscillations for at least 10 min. Synchronous Ca(2+) oscillations could be mediated by intercellular communication via gap junctions, although intercellular bridges could also be involved. We also observed an increase in [Ca(2+)]i after testosterone application, suggesting the presence of functional Sertoli cells in the SST. In summary, we believe that the SST preparation is suitable to explore the physiology of spermatogenic cells in their natural environment, within the seminiferous tubules, in particular Ca(2+) signaling phenomena, functional cell-cell communication, and multicellular functional arrangements.

A caged progesterone analog alters intracellular Ca2+ and flagellar bending in human sperm.

Progesterone is a physiological agonist for mammalian sperm, modulating its flagellar movement and facilitating the acrosome reaction. To study the initial action of progesterone, we developed a caged analog with a photosensitive group: nitrophenylethanediol, at position 20. Using this compound combined with stroboscopic illumination, we performed Ca(2)(+) imaging of human spermatozoa and analyzed the effects of progesterone on the intracellular Ca(2)(+) concentration ([Ca(2)(+)](i)) of beating flagella for the first time. We observed a transient [Ca(2)(+)](i) increase in the head and the flagellum upon photolysis of the caged progesterone and an increase in flagellar curvature. Detailed kinetic analysis revealed that progesterone elicits an increase in the [Ca(2)(+)](i) immediately in the flagellum (mid-piece and principal piece), thereafter in the head with a short time lag. This observation is different from the progesterone-induced Ca(2)(+) mobilization in mouse spermatozoa, where the Ca(2)(+) rise initiates at the base of the sperm head. Our finding is mostly consistent with the recent discovery that progesterone activates CatSper channels in human spermatozoa, but not in mouse spermatozoa.

Super-resolution microscopy for the study of store-operated calcium entry.

The use of a variety of techniques based on super-resolution (SR) microscopy unveiled a close and complex relationship between cytoskeleton reorganization and SOCE. By using SR microscopy many new proteins involved in SOCE regulation have been identified over the last few years. Many enigmas remain unsolved in this highly dynamic field, however, recent developments in SR microscopy promise new answers soon. In the present review, we describe the most relevant findings in SOCE components and SOCE modulation using different methods derived from SR microscopy.

A Machine Learning Workflow of Multiplexed Immunofluorescence Images to Interrogate Activator and Tolerogenic Profiles of Conventional Type 1 Dendritic Cells Infiltrating Melanomas of Disease-Free and Metastatic Patients.

Melanoma is the deadliest form of skin cancer. Due to its high mutation rates, melanoma is a convenient model to study antitumor immune responses. Dendritic cells (DCs) play a key role in activating cytotoxic CD8+ T lymphocytes and directing them to kill tumor cells. Although there is evidence that DCs infiltrate melanomas, information about the profile of these cells, their activity states, and potential antitumor function remains unclear, particularly for conventional DCs type 1 (cDC1). Approaches to profiling tumor-infiltrating DCs are hindered by their diversity and the high number of signals that can affect their state of activation. Multiplexed immunofluorescence (mIF) allows the simultaneous analysis of multiple markers, but image-based analysis is time-consuming and often inconsistent among analysts. In this work, we evaluated several machine learning (ML) algorithms and established a workflow of nine-parameter image analysis that allowed us to study cDC1s in a reproducible and accessible manner. Using this workflow, we compared melanoma samples between disease-free and metastatic patients at diagnosis. We observed that cDC1s are more abundant in the tumor infiltrate of the former. Furthermore, cDC1s in disease-free patients exhibit an expression profile more congruent with an activator function: CD40highPD-L1low CD86+IL-12+. Although disease-free patients were also enriched with CD40-PD-L1+ cDC1s, these cells were also more compatible with an activator phenotype. The opposite was true for metastatic patients at diagnosis who were enriched for cDC1s with a more tolerogenic phenotype (CD40lowPD-L1highCD86-IL-12-IDO+). ML-based workflows like the one developed here can be used to analyze complex phenotypes of other immune cells and can be brought to laboratories with standard expertise and computer capacity.

Extending resolution within a single imaging frame.

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.

Tuning sperm chemotaxis by calcium burst timing.

Marine invertebrate oocytes establish chemoattractant gradients that guide spermatozoa towards their source. In sea urchin spermatozoa, this relocation requires coordinated motility changes initiated by Ca(2+)-driven alterations in sperm flagellar curvature. We discovered that Lytechinus pictus spermatozoa undergo chemotaxis in response to speract, an egg-derived decapeptide previously noted to stimulate non-chemotactic motility alterations in Strongylocentrotus purpuratus spermatozoa. Sperm of both species responded to speract gradients with a sequence of turning episodes that correlate with transient flagellar Ca(2+) increases, yet only L. pictus spermatozoa accumulated at the gradient source. Detailed analysis of sperm behavior revealed that L. pictus spermatozoa selectively undergo Ca(2+) fluctuations while swimming along negative speract gradients while S. purpuratus sperm generate Ca(2+) fluctuations in a spatially non-selective manner. This difference is attributed to the selective suppression of Ca(2+) fluctuations of L. pictus spermatozoa as they swim towards the source of the chemoattractant gradient. This is the first study to compare and characterize the motility components that differ in chemotactic and non-chemotactic spermatozoa. Tuning of Ca(2+) fluctuations and associated turning episodes to the chemoattractant gradient polarity is a central feature of sea urchin sperm chemotaxis and may be a feature of sperm chemotaxis in general.

Strategies for locating the female gamete: the importance of measuring sperm trajectories in three spatial dimensions.

The spermatozoon must find its female gamete partner and deliver its genetic material to generate a new individual. This requires that the spermatozoon be motile and endowed with sophisticated swimming strategies to locate the oocyte. A common strategy is chemotaxis, in which spermatozoa detect and follow a gradient of chemical signals released by the egg and its associated structures. Decoding the female gamete's positional information is a process that spermatozoa undergo in a three-dimensional (3D) space; however, due to their speed and small size, this process has been studied almost exclusively in spermatozoa restricted to swimming in two dimensions (2D). This review examines the relationship between the mechanics of sperm propulsion and the physiological function of these cells in 3D. It also considers whether it is possible to derive all the 3D sperm swimming characteristics by extrapolating from 2D measurements. It is concluded that full insight into flagellar beat dynamics, swimming paths and chemotaxis under physiological conditions will eventually require quantitative imaging of flagellar form, ion flux changes, cell trajectories and modelling of free-swimming spermatozoa in 3D.