Biology subjects

Rickert, C.

Publications and source records attributed to Rickert, C..

5 recordsLinked to original sources

bioRxiv2024

Specific presynaptic functions require distinct Drosophila Cav2 splice isoforms

The multiplicity of neural circuits that accommodate the sheer infinite number of computations conducted by brains requires diverse synapse and neuron types. At many vertebrate synapses release probability and other aspects of presynaptic function are tuned by different combinations of Cav2.1, Cav2.2, and Cav2.3 channels. By contrast, most invertebrate genomes contain only one Cav2 gene. The one Drosophila Cav2 homolog, cacophony (cac), localizes to presynaptic active zones (AZs) to induce synaptic vesicle release. We hypothesize that Drosophila cac functional diversity is enhanced by two specific exon pairs that are mutually exclusively spliced and not conserved in vertebrates, one in the voltage sensor and one in the intracellular loop containing the binding site(s) for Ca{beta} and G-protein {beta}{gamma} subunits. We test our hypothesis by combining opto- and electrophysiological with neuroanatomical approaches at a fast glutamatergic model synapse, the Drosophila larval neuromuscular junction. We find that alternative splicing in the voltage sensor affects channel activation voltage and is imperative for normal synapse function. Only the isoform with the higher activation voltage localizes to the presynaptic AZ and mediates evoked release. Removal of these cac splice isoforms renders fast glutamatergic synapses non-functional. By contrast, alternative splicing at the other alternative exon that encodes the intracellular loop between the first and the second homologous repeats does not affect cac presynaptic AZ localization, but it tunes multiple aspects of presynaptic function. While expression of one exon yields normal transmission, expression of the other exon reduces channel number in the AZ and thus release probability. This also abolishes presynaptic homeostatic plasticity. Moreover, reduced channel number upon selective exon excision increases paired pulse ratios and the variability of synaptic depression during low frequency stimulation trains (1 and 10 Hz), and thus affects short term plasticity. Effects on short term plasticity can be rescued by increasing the external calcium concentration to match release probability to control. In sum, in Drosophila alternative splicing provides a mechanism to regulate different aspects of presynaptic functions with only one Cav2 gene.

neuroscience↗

bioRxiv2024

Caveolar Compartmentalization is Required for Stable Rhythmicity of Sinus Nodal Cells and is Disrupted in Heart Failure

BackgroundHeart rhythm relies on complex interactions between electrogenic membrane proteins and intracellular Ca2+ signaling in sinoatrial node (SAN) myocytes; however, mechanisms underlying the functional organization of proteins involved in SAN pacemaking and its structural foundation remain elusive. Caveolae are nanoscale, plasma membrane pits that compartmentalize various ion channels and transporters, including those involved in SAN pacemaking, via binding with the caveolin-3 scaffolding protein, but the precise role of caveolae in cardiac pacemaker function is unknown. Our objective was to determine the role of caveolae in SAN pacemaking and dysfunction (SND). MethodsBiochemical co-purification, in vivo electrocardiogram monitoring, ex vivo optical mapping, in vitro confocal Ca2+ imaging, and immunofluorescent and electron microscopy analyses were performed in wild type, cardiac-specific caveolin-3 knockout, and 8-weeks post-myocardial infarction heart failure (HF) mice. SAN tissue samples from donor human hearts were used for biochemical studies. We utilized a novel 3-dimensional single SAN cell mathematical model to determine the functional outcomes of protein nanodomain-specific localization and redistribution in SAN pacemaking. ResultsIn both mouse and human SANs, caveolae compartmentalized HCN4, Cav1.2, Cav1.3, Cav3.1 and NCX1 proteins within discrete pacemaker signalosomes via direct association with caveolin-3. This compartmentalization positioned electrogenic sarcolemmal proteins near the subsarcolemmal sarcoplasmic reticulum (SR) membrane and ensured fast and robust activation of NCX1 by subsarcolemmal local SR Ca2+ release events (LCRs), which diffuse across [~]15-nm subsarcolemmal cleft. Disruption of caveolae led to the development of SND via suppression of pacemaker automaticity through a 50% decrease of the L-type Ca2+ current, a negative shift of the HCN current (If) activation curve, and a 40% reduction of Na+/Ca2+-exchanger function, along with [~]2.3-times widening of the sarcolemma-SR distance. These changes significantly decreased the SAN depolarizing force, both during diastolic depolarization and upstroke phase, leading to bradycardia, sinus pauses, recurrent development of SAN quiescence, and significant increase in heart rate lability. Computational modeling, supported by biochemical studies, identified NCX1 redistribution to extra-caveolar membrane as the primary mechanism of SAN pauses and quiescence due to the impaired ability of NCX1 to be effectively activated by LCRs and trigger action potentials. HF remodeling mirrored caveolae disruption leading to NCX1-LCR uncoupling and SND. ConclusionsSAN pacemaking is driven by complex protein interactions within a nanoscale caveolar pacemaker signalosome. Disruption of caveolae leads to SND, potentially demonstrating a new dimension of SAN remodeling and providing a newly recognized target for therapy.

physiology↗

bioRxiv2024

The spatial structure of the tumor immune microenvironment can explain and predict patient response in high-grade serous carcinoma

Despite ovarian cancer being the deadliest gynecological malignancy, there has been little change to therapeutic options and mortality rates over the last three decades. Recent studies indicate that the composition of the tumor immune microenvironment (TIME) influences patient outcomes but are limited by a lack of spatial understanding. We performed multiplexed ion beam imaging (MIBI) on 83 human high-grade serous carcinoma tumors -- one of the largest protein-based, spatially-intact, single-cell resolution tumor datasets assembled -- and used statistical and machine learning approaches to connect features of the TIME spatial organization to patient outcomes. Along with traditional clinical/immunohistochemical attributes and indicators of TIME composition, we found that several features of TIME spatial organization had significant univariate correlations and/or high relative importance in high-dimensional predictive models. The top performing predictive model for patient progression-free survival (PFS) used a combination of TIME composition and spatial features. Results demonstrate the importance of spatial structure in understanding how the TIME contributes to treatment outcomes. Furthermore, the present study provides a generalizable roadmap for spatial analyses of the TIME in ovarian cancer research.

cancer biology↗

bioRxiv2022

A Platform-Independent Framework for Phenotyping of Multiplex Tissue Imaging Data

Multiplex imaging is a powerful tool to analyze the structural and functional states of cells in their morphological and pathological contexts. However, hypothesis testing with multiplex imaging data is a challenging task due to the extent and complexity of the information obtained. Various computational pipelines have been developed and validated to extract knowledge from specific imaging platforms. A common problem with customized pipelines is their reduced applicability across different imaging platforms: Every multiplex imaging technique exhibits platform-specific characteristics in terms of signal-to-noise ratio and acquisition artifacts that need to be accounted for to yield reliable and reproducible results. We propose a pixel classifier-based image preprocessing step that aims to minimize platform-dependency for all multiplex image analysis pipelines. Signal detection and noise reduction as well as artifact removal can be posed as a pixel classification problem in which all pixels in multiplex images can be assigned to two general classes of either I) signal of interest or II) artifacts and noise. The resulting feature representation maps contain pixel-accurate representations of the input data, but exhibit signifi-cantly increased signal-to-noise ratios with normalized pixel values as output data. We demonstrate the validity of our proposed image preprocessing approach by comparing the results of two well-accepted and widely-used image analysis pipelines.

bioinformatics↗

bioRxiv2022

The funny current If is essential for the fight-or-flight response in cardiac pacemaker cells

The sympathetic nervous system fight-or-flight response is characterized by a rapid increase in heart rate, which is mediated by an increase in the spontaneous action potential (AP) firing rate of pacemaker cells in the sinoatrial node. Sympathetic neurons stimulate sinoatrial myocytes (SAMs) by activating {beta} adrenergic receptors ({beta}ARs) and increasing cAMP. The funny current (If) is among the cAMP-sensitive currents in SAMs. If is critical for pacemaker activity, however, its role in the fight-or-flight response remains controversial. In this study, we used AP waveform analysis, machine learning, and dynamic clamp experiments in acutely-isolated SAMs from mice to quantitatively define the AP waveform changes and role of If in the fight-or-flight response. We found that while {beta}AR stimulation significantly altered nearly all AP waveform parameters, the increase in AP firing rate was only correlated with changes in a subset of parameters (diastolic duration, late AP duration, and diastolic depolarization rate). Dynamic clamp injection of the {beta}AR-sensitive component of If showed that it accounts for approximately 41% of the fight-or-flight increase in AP firing rate and 60% of the decrease in the interval between APs. Thus, If is an essential contributor to the fight-or-flight increase in heart rate.

physiology↗