Christopher D. Wood

Measurement of single-cell dynamics

Populations of cells are almost always heterogeneous in function and fate. To understand the plasticity of cells, it is vital to measure quantitatively and dynamically the molecular processes that underlie cell-fate decisions in single cells. Early events in cell signalling often occur within seconds of the stimulus, whereas intracellular signalling processes and transcriptional changes can take minutes or hours. By contrast, cell-fate decisions, such as whether a cell divides, differentiates or dies, can take many hours or days. Multiparameter experimental and computational methods that integrate quantitative measurement and mathematical simulation of these noisy and complex processes are required to understand the highly dynamic mechanisms that control cell plasticity and fate.

Calcium Channels and Ca2+ Fluctuations in Sperm Physiology

Generating new life in animals by sexual reproduction depends on adequate communication between mature and competent male and female gametes. Ion channels are instrumental in the dialogue between sperm, its environment, and the egg. The ability of sperm to swim to the egg and fertilize it is modulated by ion permeability changes induced by environmental cues and components of the egg outer layer. Ca(2+) is probably the key messenger in this information exchange. It is therefore not surprising that different Ca(2+)-permeable channels are distinctly localized in these tiny specialized cells. New approaches to measure sperm currents, intracellular Ca(2+), membrane potential, and intracellular pH with fluorescent probes, patch-clamp recordings, sequence information, and heterologous expression are revealing how sperm channels participate in fertilization. Certain sperm ion channels are turning out to be unique, making them attractive targets for contraception and for the discovery of novel signaling complexes.

Reactive oxygen species: A radical role in development?

Reactive oxygen species (ROS), mostly derived from mitochondrial activity, can damage various macromolecules and consequently cause cell death. This ROS activity has been characterized in vitro, and correlative evidence suggests a role in various pathological conditions. In addition to this passive ROS activity, ROS also participate in cell signaling processes, though the relevance of this function in vivo is poorly understood. Throughout development, elevated cell activity is probably accompanied by highly active metabolism and, consequently, the production of large amounts of ROS. To allow proper development, cells must protect themselves from these potentially damaging ROS. However, to what degree ROS could participate as signaling molecules controlling fundamental and developmentally relevant cellular processes such as proliferation, differentiation, and death is an open question. Here we discuss why available data do not yet provide conclusive evidence on the role of ROS in development, and we review recent methods to detect ROS in vivo and genetic strategies that can be exploited specifically to resolve these uncertainties.

Real-time analysis of the role of Ca2+ in flagellar movement and motility in single sea urchin sperm

Eggs of many marine and mammalian species attract sperm by releasing chemoattractants that modify the bending properties of flagella to redirect sperm paths toward the egg. This process, called chemotaxis, is dependent on extracellular Ca2+. We used stroboscopic fluorescence imaging to measure intracellular Ca2+ concentration ([Ca2+]i) in the flagella of swimming sea urchin sperm. Uncaging of cyclic GMP induced Ca2+ entry via at least two distinct pathways, and we identified a nimodipine-sensitive pathway, compartmentalized in the flagella, as a key regulator of flagellar bending and directed motility changes. We found that, contrary to current models, the degree of flagellar bending does not vary in proportion to the overall [Ca2+]i. Instead we propose a new model whereby flagella bending is increased by Ca2+ flux through the nimodipine-sensitive pathway, and is unaffected by [Ca2+]i increases through alternative pathways.

Speract induces calcium oscillations in the sperm tail

Sea urchin sperm motility is modulated by sperm-activating peptides. One such peptide, speract, induces changes in intracellular free calcium concentration ([Ca2+]i). High resolution imaging of single sperm reveals that speract-induced changes in [Ca2+]i have a complex spatiotemporal structure. [Ca2+]i increases arise in the tail as periodic oscillations; [Ca2+]i increases in the sperm head lag those in the tail and appear to result from the summation of the tail signal transduction events. The period depends on speract concentration. Infrequent spontaneous [Ca2+]i transients were also seen in the tail of unstimulated sperm, again with the head lagging the tail. Speract-induced fluctuations were sensitive to membrane potential and calcium channel blockers, and were potentiated by niflumic acid, an anion channel blocker. 3-isobutyl-1-methylxanthine, which potentiates the cGMP/cAMP-signaling pathways, abolished the [Ca2+]i fluctuations in the tail, leading to a very delayed and sustained [Ca2+]i increase in the head. These data point to a model in which a messenger generated periodically in the tail diffuses to the head. Sperm are highly polarized cells. Our results indicate that a clear understanding of the link between [Ca2+]i and sperm motility will only be gained by analysis of [Ca2+]i signals at the level of the single sperm.

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.

Sperm-activating peptides in the regulation of ion fluxes, signal transduction and motility

Echinoderm sperm use cyclic nucleotides (CNs) as essential second messengers to locate and swim towards the egg. Sea urchin sperm constitute a rich source of membrane-bound guanylyl cyclase (mGC), which was first cloned from sea urchin testis by the group of David Garbers. His group also identified speract, the first sperm-activating peptide (SAP) to be isolated from the egg investment (or egg jelly). This decapeptide stimulates sperm mGC causing a fast transient increase in cGMP that triggers an orchestrated set of physiological responses including: changes in: membrane potential, intracellular pH (pHi), intracellular Ca2+ concentration ([Ca2+]i) and cAMP levels. Evidence from several groups indicated that cGMP activation of a K+ selective channel was the first ion permeability change in the signaling cascade induced by SAPs, and recently the candidate gene was finally identified. Each of the 4 repeated, 6 trans-membrane segments of this channel contains a cyclic nucleotide binding domain. Together they comprise a single polypeptide chain like voltage-gated Na+ or Ca2+ channels. This new type of channel, named tetraKCNG, appears to belong to the exclusive club of novel protein families expressed only in sperm and its progenitors. SAPs also induce fluctuations in flagellar [Ca2+]i that correlate with changes in flagellar form and regulate sperm trajectory. The motility changes depend on [Ca2+]i influx through specific Ca2+ channels and not on the overall [Ca2+]i in the sperm flagellum. All cilia and flagella have a conserved axonemal structure and thus understanding how Ca2+ regulates cilia and flagella beating is a fundamental question.

Tracking sperm in three-dimensions.

Sperm motility, crucial for fertilization, has been mostly studied in two dimensions (2D) by recording their swimming trajectories near a flat surface. However, spermatozoa swim in three-dimensions (3D) to find eggs, with their speed being the main impediment to track them under realistic conditions. Here, we describe a novel method allowing 3D tracking and analysis of the trajectories of multiple free-swimming sperm. The system uses a piezo-electric device displacing a large focal distance objective mounted on a microscope to acquire 70 image stacks per second, each stack composed of 60 images that span a depth of 100 microm. With this method, 3D paths of multiple sperm in the same field could be visualized simultaneously during 1 s. Within the same sample we found that surface-confined sperm swam 25% slower, produced 3-fold fewer circular revolutions per second, and had trajectories of 134% greater radius of curvature than those sperm swimming freely in 3D.

Stroboscopic illumination using light-emitting diodes reduces phototoxicity in fluorescence cell imaging

Excited fluorophores produce reactive oxygen species that are toxic toward many live cells (phototoxicity) and accelerate bleaching of the fluorophores during the course of extended or repeated measurements (photobleaching). We recently developed an illumination system for fluorescence microscopy using a high power light-emitting diode (LED), which can emit short pulses of light (0.5-2 ms) to excite fluorophores. This system minimizes illumination time, thus reducing phototoxicity and photobleaching artifacts. To demonstrate the usefulness of the new system, we compared images of human sperm loaded with various fluorescent indicators and excited with either a conventional mercury lamp as a continuous excitation light source or the LED as a source of pulsed illumination. We found that sperm motility decreased rapidly and photobleaching was relatively rapid under continuous illumination, whereas under pulsed LED illumination, motility was maintained and photobleaching was much reduced. Therefore, fluorescence microscopy using LED-based pulsed illumination offers significant advantages for long-term live cell imaging, reducing the degree of phototoxicity, and extending the effective lifetime of fluorophores.

Real-time imaging of gene expression in single living cells

Recent advances in reporter gene technologies are now allowing us to image gene transcription at the single cell level, using either fluorescence or luminescence microscopy. Here, the basis of these techniques is outlined and their advantages and disadvantages in various biological systems are discussed.