Dual role of valosin-containing protein (VCP/p97) in mouse sperm during capacitation.

Valosin-containing protein (VCP; aka p97), a member of the AAA (ATPases Associated with various cellular Activities) family, has been associated with a wide range of cellular functions. While previous evidence has shown its presence in mammalian sperm, our study unveils its function in mouse sperm. Notably, we found that mouse VCP does not undergo tyrosine phosphorylation during capacitation and exhibits distinct localization patterns. In the sperm head, it resides within the equatorial segment and, following acrosomal exocytosis, it is released and cleaved. In the flagellum, VCP is observed in the principal and midpiece. Furthermore, our research highlights a unique role for VCP in the cAMP/PKA pathway during capacitation. Pharmacological inhibition of sperm VCP led to reduced intracellular cAMP levels that resulted in decreased phosphorylation in PKA substrates and tyrosine residues and diminished fertilization competence. Our results show that in mouse sperm, VCP plays a pivotal role in regulating cAMP production, probably by the modulation of soluble adenylyl cyclase activity.

Cdc42 localized in the CatSper signaling complex regulates cAMP-dependent pathways in mouse sperm.

Sperm acquire the ability to fertilize in a process called capacitation and undergo hyperactivation, a change in the motility pattern, which depends on Ca2+ transport by CatSper channels. CatSper is essential for fertilization and it is subjected to a complex regulation that is not fully understood. Here, we report that similar to CatSper, Cdc42 distribution in the principal piece is confined to four linear domains and this localization is disrupted in CatSper1-null sperm. Cdc42 inhibition impaired CatSper activity and other Ca2+ -dependent downstream events resulting in a severe compromise of the sperm fertilizing potential. We also demonstrate that Cdc42 is essential for CatSper function by modulating cAMP production by soluble adenylate cyclase (sAC), providing a new regulatory mechanism for the stimulation of CatSper by the cAMP-dependent pathway. These results reveal a broad mechanistic insight into the regulation of Ca2+ in mammalian sperm, a matter of critical importance in male infertility as well as in contraception.

Multifractional Brownian motion characterization based on Hurst exponent estimation and statistical learning.

This paper proposes an approach for the estimation of a time-varying Hurst exponent to allow accurate identification of multifractional Brownian motion (MFBM). The contribution provides a prescription for how to deal with the MFBM measurement data to solve regression and classification problems. Theoretical studies are supplemented with computer simulations and real-world examples. Those prove that the procedure proposed in this paper outperforms the best-in-class algorithm.

The actin cytoskeleton of the mouse sperm flagellum is organized in a helical structure.

Conception in mammals is determined by the fusion of a sperm cell with an oocyte during fertilization. Motility is one of the features of sperm that allows them to succeed in fertilization, and their flagellum is essential for this function. Longitudinally, the flagellum can be divided into the midpiece, the principal piece and the end piece. A precise cytoskeletal architecture of the sperm tail is key for the acquisition of fertilization competence. It has been proposed that the actin cytoskeleton plays essential roles in the regulation of sperm motility; however, the actin organization in sperm remains elusive. In the present work, we show that there are different types of actin structures in the sperm tail by using three-dimensional stochastic optical reconstruction microscopy (STORM). In the principal piece, actin is radially distributed between the axoneme and the plasma membrane. The actin-associated proteins spectrin and adducin are also found in these structures. Strikingly, polymerized actin in the midpiece forms a double-helix that accompanies mitochondria. Our findings illustrate a novel specialized structure of actin filaments in a mammalian cell.

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.

Elucidating the Origin of Heterogeneous Anomalous Diffusion in the Cytoplasm of Mammalian Cells.

Diffusion of tracer particles in the cytoplasm of mammalian cells is often anomalous with a marked heterogeneity even within individual particle trajectories. Despite considerable efforts, the mechanisms behind these observations have remained largely elusive. To tackle this problem, we performed extensive single-particle tracking experiments on quantum dots in the cytoplasm of living mammalian cells at varying conditions. Analyses of the trajectories reveal a strong, microtubule-dependent subdiffusion with antipersistent increments and a substantial heterogeneity. Furthermore, particles stochastically switch between different mobility states, most likely due to transient associations with the cytoskeleton-shaken endoplasmic reticulum network. Comparison to simulations highlight that all experimental observations can be fully described by an intermittent fractional Brownian motion, alternating between two states of different mobility.

Anomalous protein kinetics on low-fouling surfaces.

In this work, protein-surface interactions were probed in terms of adsorption and desorption of a model protein, bovine serum albumin, on a low-fouling surface with single-molecule localization microscopy. Single-molecule experiments enable precise determination of both adsorption and desorption rates. Strikingly the experimental data show anomalous desorption kinetics, evident as a surface dwell time that exhibits a power-law distribution, i.e. a heavy-tailed rather than the expected exponential distribution. As a direct consequence of this heavy-tailed distribution, the average desorption rate depends upon the time scale of the experiment and the protein surface concentration does not reach equilibrium. Further analysis reveals that the observed anomalous desorption emerges due to the reversible formation of a small fraction of soluble protein multimers (small oligomers), such that each one desorbs from the surface with a different rate. The overall kinetics can be described by a series of elementary reactions, yielding simple scaling relations that predict experimental observations. This work reveals a mechanistic origin for anomalous desorption kinetics that can be employed to interpret observations where low-protein fouling surfaces eventually foul when in long-term contact with protein solutions. The work also provides new insights that can be used to define design principles for non-fouling surfaces and to predict their performance.

Sperm Differentiation: The Role of Trafficking of Proteins.

Sperm differentiation encompasses a complex sequence of morphological changes that takes place in the seminiferous epithelium. In this process, haploid round spermatids undergo substantial structural and functional alterations, resulting in highly polarized sperm. Hallmark changes during the differentiation process include the formation of new organelles, chromatin condensation and nuclear shaping, elimination of residual cytoplasm, and assembly of the sperm flagella. To achieve these transformations, spermatids have unique mechanisms for protein trafficking that operate in a coordinated fashion. Microtubules and filaments of actin are the main tracks used to facilitate the transport mechanisms, assisted by motor and non-motor proteins, for delivery of vesicular and non-vesicular cargos to specific sites. This review integrates recent findings regarding the role of protein trafficking in sperm differentiation. Although a complete characterization of the interactome of proteins involved in these temporal and spatial processes is not yet known, we propose a model based on the current literature as a framework for future investigations.

Disruption of protein kinase A localization induces acrosomal exocytosis in capacitated mouse sperm.

Protein kinase A (PKA) is a broad-spectrum Ser/Thr kinase involved in the regulation of several cellular activities. Thus, its precise activation relies on being localized at specific subcellular places known as discrete PKA signalosomes. A-Kinase anchoring proteins (AKAPs) form scaffolding assemblies that play a pivotal role in PKA regulation by restricting its activity to specific microdomains. Because one of the first signaling events observed during mammalian sperm capacitation is PKA activation, understanding how PKA activity is restricted in space and time is crucial to decipher the critical steps of sperm capacitation. Here, we demonstrate that the anchoring of PKA to AKAP is not only necessary but also actively regulated during sperm capacitation. However, we find that once capacitated, the release of PKA from AKAP promotes a sudden Ca2+ influx through the sperm-specific Ca2+ channel CatSper, starting a tail-to-head Ca2+ propagation that triggers the acrosome reaction. Three-dimensional super-resolution imaging confirmed a redistribution of PKA within the flagellar structure throughout the capacitation process, which depends on anchoring to AKAP. These results represent a new signaling event that involves CatSper Ca2+ channels in the acrosome reaction, sensitive to PKA stimulation upon release from AKAP.

The tyrosine kinase FER is responsible for the capacitation-associated increase in tyrosine phosphorylation in murine sperm.

Sperm capacitation is required for fertilization. At the molecular level, this process is associated with fast activation of protein kinase A. Downstream of this event, capacitating conditions lead to an increase in tyrosine phosphorylation. The identity of the tyrosine kinase(s) mediating this process has not been conclusively demonstrated. Recent experiments using stallion and human sperm have suggested a role for PYK2 based on the use of small molecule inhibitors directed against this kinase. However, crucially, loss-of-function experiments have not been reported. Here, we used both pharmacological inhibitors and genetically modified mice models to investigate the identity of the tyrosine kinase(s) mediating the increase in tyrosine phosphorylation in mouse sperm. Similar to stallion and human, PF431396 blocks the capacitation-associated increase in tyrosine phosphorylation. Yet, sperm from Pyk2(-/-) mice displayed a normal increase in tyrosine phosphorylation, implying that PYK2 is not responsible for this phosphorylation process. Here, we show that PF431396 can also inhibit FER, a tyrosine kinase known to be present in sperm. Sperm from mice targeted with a kinase-inactivating mutation in Fer failed to undergo capacitation-associated increases in tyrosine phosphorylation. Although these mice are fertile, their sperm displayed a reduced ability to fertilize metaphase II-arrested eggs in vitro.

Steady-state reaction rate of diffusion-controlled reactions in sheets.

In many biological situations, a species arriving from a remote source diffuses in a domain confined between two parallel surfaces until it finds a binding partner. Since such a geometric shape falls in between two- and three-dimensional settings, the behavior of the macroscopic reaction rate and its dependence on geometric parameters are not yet understood. Modeling the geometric setup by a capped cylinder with a concentric disk-like reactive region on one of the lateral surfaces, we provide an exact semi-analytical solution of the steady-state diffusion equation and compute the diffusive flux onto the reactive region. We explore the dependence of the macroscopic reaction rate on the geometric parameters and derive asymptotic results in several limits. Using the self-consistent approximation, we also obtain a simple fully explicit formula for the reaction rate that exhibits a transition from two-dimensional to three-dimensional behavior as the separation distance between lateral surfaces increases. Biological implications of these results are discussed.

Induction of stable ER-plasma-membrane junctions by Kv2.1 potassium channels.

Junctions between cortical endoplasmic reticulum (cER) and the plasma membrane are a subtle but ubiquitous feature in mammalian cells; however, very little is known about the functions and molecular interactions that are associated with neuronal ER-plasma-membrane junctions. Here, we report that Kv2.1 (also known as KCNB1), the primary delayed-rectifier K(+) channel in the mammalian brain, induces the formation of ER-plasma-membrane junctions. Kv2.1 localizes to dense, cell-surface clusters that contain non-conducting channels, indicating that they have a function that is unrelated to membrane-potential regulation. Accordingly, Kv2.1 clusters function as membrane-trafficking hubs, providing platforms for delivery and retrieval of multiple membrane proteins. Using both total internal reflection fluorescence and electron microscopy we demonstrate that the clustered Kv2.1 plays a direct structural role in the induction of stable ER-plasma-membrane junctions in both transfected HEK 293 cells and cultured hippocampal neurons. Glutamate exposure results in a loss of Kv2.1 clusters in neurons and subsequent retraction of the cER from the plasma membrane. We propose Kv2.1-induced ER-plasma-membrane junctions represent a new macromolecular plasma-membrane complex that is sensitive to excitotoxic insult and functions as a scaffolding site for both membrane trafficking and Ca(2+) signaling.

Single-Molecule Imaging of Nav1.6 on the Surface of Hippocampal Neurons Reveals Somatic Nanoclusters.

Voltage-gated sodium (Nav) channels are responsible for the depolarizing phase of the action potential in most nerve cells, and Nav channel localization to the axon initial segment is vital to action potential initiation. Nav channels in the soma play a role in the transfer of axonal output information to the rest of the neuron and in synaptic plasticity, although little is known about Nav channel localization and dynamics within this neuronal compartment. This study uses single-particle tracking and photoactivation localization microscopy to analyze cell-surface Nav1.6 within the soma of cultured hippocampal neurons. Mean-square displacement analysis of individual trajectories indicated that half of the somatic Nav1.6 channels localized to stable nanoclusters ∼230 nm in diameter. Strikingly, these domains were stabilized at specific sites on the cell membrane for >30 min, notably via an ankyrin-independent mechanism, indicating that the means by which Nav1.6 nanoclusters are maintained in the soma is biologically different from axonal localization. Nonclustered Nav1.6 channels showed anomalous diffusion, as determined by mean-square-displacement analysis. High-density single-particle tracking of Nav channels labeled with photoactivatable fluorophores in combination with Bayesian inference analysis was employed to characterize the surface nanoclusters. A subpopulation of mobile Nav1.6 was observed to be transiently trapped in the nanoclusters. Somatic Nav1.6 nanoclusters represent a new, to our knowledge, type of Nav channel localization, and are hypothesized to be sites of localized channel regulation.

Strange kinetics of bulk-mediated diffusion on lipid bilayers.

Diffusion at solid-liquid interfaces is crucial in many technological and biophysical processes. Although its behavior seems to be deceivingly simple, recent studies showing passive superdiffusive transport suggest that diffusion on surfaces may hide rich complexities. In particular, bulk-mediated diffusion occurs when molecules are transiently released from the surface to perform three-dimensional excursions into the liquid bulk. This phenomenon bears the dichotomy where a molecule always return to the surface but the mean jump length is infinite. Such behavior is associated with a breakdown of the central limit theorem and weak ergodicity breaking. Here, we use single-particle tracking to study the statistics of bulk-mediated diffusion on a supported lipid bilayer. We find that the time-averaged mean square displacement (MSD) of individual trajectories, the archetypal measure in diffusion processes, does not converge to the ensemble MSD but it remains a random variable, even in the long observation-time limit. The distribution of time averages is shown to agree with a Lévy flight model. Our results also unravel intriguing anomalies in the statistics of displacements. The time-averaged MSD is shown to depend on experimental time and investigations of fractional moments show a scaling 〈|r(t)|(q)〉∼t(qν(q)) with non-linear exponents, i.e. ν(q) ≠ const. This type of behavior is termed strong anomalous diffusion and is rare among experimental observations.

Quantifying the dynamic interactions between a clathrin-coated pit and cargo molecules.

Clathrin-mediated endocytosis takes place through the recruitment of cargo molecules into a growing clathrin-coated pit (CCP). Despite the importance of this process to all mammalian cells, little is yet known about the interaction dynamics between cargo and CCPs. These interactions are difficult to study because CCPs display a large degree of lifetime heterogeneity and the interactions with cargo molecules are time dependent. We use single-molecule total internal reflection fluorescence microscopy, in combination with automatic detection and tracking algorithms, to directly visualize the recruitment of individual voltage-gated potassium channels into forming CCPs in living cells. We observe association and dissociation of individual channels with a CCP and, occasionally, their internalization. Contrary to widespread ideas, cargo often escapes from a pit before abortive CCP termination or endocytic vesicle production. Thus, the binding times of cargo molecules associating to CCPs are much shorter than the overall endocytic process. By measuring tens of thousands of capturing events, we build the distribution of capture times and the times that cargo remains confined to a CCP. An analytical stochastic model is developed and compared with the measured distributions. Due to the dynamic nature of the pit, the model is non-Markovian and it displays long-tail power law statistics. The measured distributions and model predictions are in excellent agreement over more than five orders of magnitude. Our findings identify one source of the large heterogeneities in CCP dynamics and provide a mechanism for the anomalous diffusion of proteins in the plasma membrane.

Mechanisms underlying anomalous diffusion in the plasma membrane.

The plasma membrane is a complex fluid where lipids and proteins undergo diffusive motion critical to biochemical reactions. Through quantitative imaging analyses such as single-particle tracking, it is observed that diffusion in the cell membrane is usually anomalous in the sense that the mean squared displacement is not linear with time. This chapter describes the different models that are employed to describe anomalous diffusion, paying special attention to the experimental evidence that supports these models in the plasma membrane. We review models based on anticorrelated displacements, such as fractional Brownian motion and obstructed diffusion, and nonstationary models such as continuous time random walks. We also emphasize evidence for the formation of distinct compartments that transiently form on the cell surface. Finally, we overview heterogeneous diffusion processes in the plasma membrane, which have recently attracted considerable interest.

Ergodic and nonergodic processes coexist in the plasma membrane as observed by single-molecule tracking.

Diffusion in the plasma membrane of living cells is often found to display anomalous dynamics. However, the mechanism underlying this diffusion pattern remains highly controversial. Here, we study the physical mechanism underlying Kv2.1 potassium channel anomalous dynamics using single-molecule tracking. Our analysis includes both time series of individual trajectories and ensemble averages. We show that an ergodic and a nonergodic process coexist in the plasma membrane. The ergodic process resembles a fractal structure with its origin in macromolecular crowding in the cell membrane. The nonergodic process is found to be regulated by transient binding to the actin cytoskeleton and can be accurately modeled by a continuous-time random walk. When the cell is treated with drugs that inhibit actin polymerization, the diffusion pattern of Kv2.1 channels recovers ergodicity. However, the fractal structure that induces anomalous diffusion remains unaltered. These results have direct implications on the regulation of membrane receptor trafficking and signaling.

GLT-1a glutamate transporter nanocluster localization is associated with astrocytic actin and neuronal Kv2 clusters at sites of neuron-astrocyte contact.

Introduction: Astrocytic GLT-1 glutamate transporters ensure the fidelity of glutamic neurotransmission by spatially and temporally limiting glutamate signals. The ability to limit neuronal hyperactivity relies on the localization and diffusion of GLT-1 on the astrocytic surface, however, little is known about the underlying mechanisms. We show that two isoforms of GLT-1, GLT-1a and GLT-1b, form nanoclusters on the surface of transfected astrocytes and HEK-293 cells. Methods: We used both fixed and live cell super-resolution imaging of fluorescent protein and epitope tagged proteins in co-cultures of rat astrocytes and neurons. Immunofluorescence techniques were also used. GLT1 diffusion was assessed via single particle tracking and fluorescence recovery after photobleach (FRAP). Results: We found GLT-1a, but not GLT-1b, nanoclusters concentrated adjacent to actin filaments which was maintained after addition of glutamate. GLT-1a nanocluster concentration near actin filaments was prevented by expression of a cytosolic GLT-1a C-terminus, suggesting the C-terminus is involved in the localization adjacent to cortical actin. Using super-resolution imaging, we show that astrocytic GLT-1a and actin co-localize in net-like structures around neuronal Kv2.1 clusters at points of neuron/astrocyte contact. Conclusion: Overall, these data describe a novel relationship between GLT-1a and cortical actin filaments, which localizes GLT-1a near neuronal structures responsive to ischemic insult.

A versatile kinase mobility shift assay (KiMSA) for PKA analysis and cyclic AMP detection in sperm physiology (and beyond).

The cAMP-dependent protein kinase (PKA) is one of the most extensively distributed kinases among intracellular signal cascades, with a pivotal role in the regulation of various processes, including the capacitation of sperm cells. Traditional assessments of PKA activity relies on the utilization of [γ-32P] ATP and the Kemptide substrate. This methodology presents several major drawbacks, including high-costs and health risks derived from the manipulation of radioactive isotopes. In this work we introduce an enhanced non-radioactive assay for quantifying PKA activity, termed KiMSA which relies on the use of a fluorescent-labeled Kemptide (Kemptide-FITC). Once the kinase reaction is terminated, the products can be easily resolved through electrophoresis on an agarose gel and quantified by fluorescence densitometry. We show that the KiMSA assay is suitable for purified PKA, and also to address both basal and capacitation induced PKA activity in mouse sperm cells. Furthermore, the assay enables monitoring the inhibition of PKA with inhibitors such as sPKI and H-89 in live cells. Therefore, the experimental and optimal assay conditions are set so that the KiMSA assay can be used to either assess in vitro as well as in vivo PKA activity in sperm cells. Finally, this method allows for measurement of cAMP concentrations, rendering a versatile technique for the study of cAMP/PKA pathways.

The sodium-proton exchangers sNHE and NHE1 control plasma membrane hyperpolarization in mouse sperm.

Sperm capacitation, crucial for fertilization, occurs in the female reproductive tract and can be replicated in vitro using a medium rich in bicarbonate, calcium, and albumin. These components trigger the cAMP-PKA signaling cascade, proposed to promote hyperpolarization of the mouse sperm plasma membrane through activation of SLO3 K+ channel. Hyperpolarization is a hallmark of capacitation: proper membrane hyperpolarization renders higher in vitro fertilizing ability, while Slo3 KO mice are infertile. However, the precise regulation of SLO3 opening remains elusive. Our study challenges the involvement of PKA in this event and reveals the role of Na+/H+ exchangers. During capacitation, calcium increase through CatSper channels activates NHE1, while cAMP directly stimulates the sperm-specific NHE, collectively promoting the alkalinization threshold needed for SLO3 opening. Hyperpolarization then feeds back Na+/H+ activity. Our work is supported by pharmacology, and a plethora of KO mouse models, and proposes a novel pathway leading to hyperpolarization.