Biology subjects

Jorgensen, E. M.

Publications and source records attributed to Jorgensen, E. M..

13 recordsLinked to original sources

bioRxiv2026

Modular gene tagging in C. elegans

Tagging a gene endogenously can identify when the gene is expressed and where the protein is localized. CRISPR is the primary tool for generating tags of endogenous genes, but it is error-prone and requires unique reagents for each gene and tag. Recombinases can insert DNA in an error-free and modular manner. Here, we tested eight recombinases for germline function in the nematode C. elegans, and introduce PhIT, a recombinase-based method for protein tagging. First, a short 39bp PhiC31 attB landing pad is inserted into the locus by CRISPR. This strain is a resource which can be used to insert a variety of modular tags. Second, tags are inserted by the integrase PhiC31, and in tandem, extraneous backbone sequences are removed by a tyrosine recombinase. Current modular tags include seven different fluorescent proteins, FLP-regulated cell-specific expression constructs, and degron tags. Importantly, tags can be inserted by genetic crosses instead of by microinjection.

genomics↗

bioRxiv2025

High-efficiency targeted integration of extrachromosomal arrays in C. elegans using PhiC31 integrase

Extrachromosomal arrays are unique chromosome-like structures created from DNA injected into the C. elegans germline. However, they are unstable unless integrated into a chromosome. Current methods for integration using X-rays or CRISPR can damage DNA and exhibit low efficiency. We demonstrate that the viral integrase PhiC31, which mediates non-mutagenic recombination between short attB and attP sequences, can be used for extremely efficient and targeted integration of arrays. Arrays were integrated by PhIAT (PhiC31-mediated Integration of Arrays of Transgenes) at attB sites on three chromosomes, including at a fluorescent landing pad. Moreover, integrations can be inserted at any arbitrary site in the genome by simultaneously co-injecting Cas9 RNP, an attB repair template, and the DNA components for the array - thereby providing sites on all six chromosomes. Single injections can integrate arrays ranging in size from 1 to 18 megabases. PhIAT makes it practical to study genomes of other organisms in the nematode; one of our strains incorporates 65% of the yeast genome at a single site in the worm genome. PhIAT will accelerate a shift from unstable extrachromosomal arrays to direct integration of arrays in C. elegans.

genetics↗

bioRxiv2025

The neuron-intrinsic membrane skeleton is required for motor neuron integrity throughout lifespan

Axons experience physical stress throughout an organisms lifetime, and disruptions in axonal integrity are hallmarks of both neurodegenerative diseases and traumatic injuries. The spectrin-based membrane periodic skeleton (MPS) is proposed to have a crucial role in maintaining axonal strength, flexibility, and resilience. To investigate the importance of the intrinsic MPS for GABAergic motor neuron integrity in C. elegans, we employed the auxin-inducible degron system to degrade {beta}-spectrin/UNC-70 in a cell-type specific and time-dependent manner. Degradation of {beta}-spectrin from all neurons beginning at larval development resulted in widespread axon breakage and regeneration in VD/DD GABAergic motor neurons in both larval and adult animals. Similarly, targeted degradation of {beta}-spectrin in GABA neurons alone resulted in extensive breakage. Moreover, we found that depleting {beta}-spectrin from the mature nervous system also induced axon breaks. By contrast, epidermal {beta}-spectrin was not required for axon integrity of VD/DD neurons. These findings demonstrate the cell-intrinsic importance of neuronal {beta}-spectrin/UNC-70 for axon integrity both during development and in adulthood.

neuroscience↗

bioRxiv2023

Rebalancing the motor circuit restores movement in a Caenorhabditis elegans model for TDP-43-toxicity

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia are caused by the abnormal accumulation of TAR DNA-binding protein 43 (TDP-43) in the cytoplasm of neurons. How TDP-43 accumulation leads to disease symptoms is not well-characterized. Here, we use a C. elegans model for TDP-43-induced toxicity to identify the biological mechanisms that lead to disease-related phenotypes. By applying deep behavioral phenotyping, we established a phenotypic fingerprint of TDP-43 worms. This fingerprint was compared to that of 294 C. elegans mutants, in which genes were mutated that are important for nervous system and muscle functioning. By using a computational clustering approach, we found that the release of acetylcholine and GABA was the primary defect in TDP-43 worms. We then functionally dissected the neuromuscular circuit to show that GABA transmission was more severely diminished compared to acetylcholine. Whereas the loss of GABA transmission was caused by a profound loss of GABA synapses, acetylcholine neurons appeared to be functionally silenced. Enhancing functional output of repressed acetylcholine neurons at the level of G-protein coupled receptors or through optogenetic stimulation restored neurotransmission, but inefficiently rescued locomotion. Surprisingly, rebalancing the excitatory and inhibitory input by simultaneous stimulation of GABA and acetylcholine input into muscles not only synergized the effects of boosting individual neurotransmitter systems, but instantaneously improved movement. Our results suggest that interventions accounting for the altered connectome may be more efficient in restoring motor function than those solely focusing on diseased neuron populations.

cell biology↗

bioRxiv2022

A Glass Phase Plate for Wavelength SensitiveSuperresolution Microscopy

Multicolor localization microscopy typically relies on sequential imaging and bandpass filters to distinguish fluorescent tags, which introduces temporal delays during live imaging, and decreases photon yield. By engineering the point-spread function (PSF), different fluors can be imaged simultaneously and distinguished by their unique patterns, without discarding photons. Here, we insert a silicon-dioxide phase plate at the Fourier plane of the detection path of a wide-field fluorescence microscope to produce distinguishable PSFs (X-PSFs) at different wavelengths. We demonstrate that the resulting PSFs can be localized spatially and spectrally using a statistics-based computational algorithm and can be utilized for hyper-spectral super-resolution microscopy of biological samples. Single PSFs in fixed U2OS cells were acquired using dSTORM with simultaneous illumination of fluors without emission filters. The modified PSF achieves [~]21 nm lateral localization precision (FWHM), [~]17 nm axial precision (FWHM) with an average of 1,800 - 3,500 photons per PSF and a background as high as 130 - 400 photons per pixel. The modified PSF can distinguish up to three fluorescent probes with [~]80 nm peak-to-peak separation between consecutive spectra.

biophysics↗

bioRxiv2022

CaV1 and CaV2 calcium channels mediate the release of distinct pools of synaptic vesicles

Activation of voltage-gated calcium channels at synapses leads to local increases in calcium and the fusion of synaptic vesicles. However, presynaptic output will be determined by the density of calcium channels, the dynamic properties of the channel, the distance to docked vesicles, and the release probability at the docking site. We demonstrate that at C. elegans neuromuscular junctions two different classes of voltage-gated calcium channels, CaV2 and CaV1, mediate the release of distinct pools of synaptic vesicles. CaV2 channels are concentrated in densely packed clusters [~]300 nm in diameter with the active zone proteins Neurexin, -Liprin, SYDE, ELKS/CAST, RIM-BP, -Catulin, and MAGI1. CaV2 channels mediate the fusion of vesicles docked adjacent to the dense projection and are colocalized with the synaptic vesicle priming protein UNC-13L. By contrast, CaV1 channels are dispersed in the synaptic varicosity and are coupled to internal calcium stores via the ryanodine receptor. CaV1 and ryanodine receptor mediate the fusion of vesicles docked broadly in the synaptic varicosity and are colocalized with the vesicle priming protein UNC-13S. Distinct synaptic vesicle pools, released by different calcium channels, could be used to tune the speed, voltage-dependence, and quantal content of neurotransmitter release.

neuroscience↗

bioRxiv2022

Syntaxin Habc is required to open Unc18 to template SNARE assembly

SNARE and Unc18 proteins form the core of the membrane fusion complex at synapses. The fusion machinery is evolutionarily ancient and mediates constitutive fusion in yeast. We demonstrate that the SNARE and Unc18 machinery in the nematode C. elegans can be replaced by yeast proteins and still carry out synaptic transmission. However, substitutions of individual components from yeast disrupts fusion. To understand the functional interactions within the core machinery we adopted an interspecies complementation approach using yeast. Synaptic transmission could be restored in chimeras when two key interfaces were present: a novel Habc-Unc18 contact site and an Unc18-SNARE motif contact site. An open form of Unc18 could bypass the requirement for the Habc-Unc18 interface. Together, these data suggest that the Habc domain of syntaxin is required for Unc18 to adopt an open conformation; open Unc18 then templates SNARE complex formation.

neuroscience↗

bioRxiv2021

High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome

Gene editing in C. elegans using plasmid-based CRISPR reagents requires microinjection of many animals to produce a single edit. Germline silencing of plasmid-borne Cas9 is a major cause of inefficient editing. Here, we present a set of C. elegans strains that constitutively express Cas9 in the germline from an integrated transgene. These strains markedly improve the success rate for plasmid-based CRISPR edits. For simple GFP insertions, 60 - 100% of injected animals typically produce edited progeny, depending on the target locus. Template-guided editing from an extrachromosomal array is maintained over multiple generations. We have built strains with the Cas9 transgene on multiple chromosomes. Additionally, each Cas9 locus also contains a heatshock-driven Cre recombinase for selectable marker removal and a bright fluorescence marker for easy outcrossing. These integrated Cas9 strains greatly reduce the workload for producing individual genome edits. Author SummaryGermlines have evolved specialized mechanisms to protect themselves from invasions by transposons and viruses, which create barriers to genome editing techniques. For example, transgenes are silenced in the germline of the nematode C. elegans, thereby creating a barrier to CRISPR editing by Cas9. To facilitate gene editing, we built a collection of C. elegans strains in which Cas9 is never silenced. CRISPR is significantly more efficient in these animals, decreasing the effort researchers need to expend to get edited animals. The strains are available in multiple genetic backgrounds, and contain accessory transgenes to simplify downstream genetics. Together, these strains enable efficient, low-cost genome editing in C. elegans.

genetics↗

bioRxiv2021

Dynamin is primed at endocytic sites for ultrafast endocytosis

Dynamin mediates fission of vesicles from the plasma membrane during endocytosis. Typically, dynamin is recruited from the cytosol to endocytic sites, requiring seconds to tens of seconds. However, ultrafast endocytosis in neurons internalizes vesicles as quickly as 50 ms during synaptic vesicle recycling. Here we demonstrate that Dynamin 1 is pre-recruited to endocytic sites for ultrafast endocytosis. Specifically, Dynamin 1xA, a splice variant of Dynamin 1, interacts with Syndapin 1 to form molecular condensates on the plasma membrane when the proline-rich domain of this variant is dephosphorylated. When this domain is mutated to include phosphomimetic residues or Syndapin 1s dynamin-interacting domain is mutated, Dynamin 1xA becomes diffuse, and consequently, ultrafast endocytosis slows down by [~]100-fold. Mechanistically, Syndapin 1 acts as an adaptor by binding the plasma membrane and stores Dynamin 1xA at endocytic sites. This cache bypasses the recruitment step and accelerates endocytosis at synapses.

neuroscience↗

bioRxiv2020

SynapsEM: computer-assisted synapse morphometry

The structural features of a synapse, in part, determine its output. Synapses are extremely small and tightly packed with vesicles and other organelles. Visualizing synaptic structure requires imaging by electron microscopy, and the features in micrographs must be quantified using morphometry. Three parameters are typically assessed from each specimen: 1) the sizes of individual vesicles and organelles, 2) the absolute number and densities of organelles, and 3) distances between organelles and key features at synapses such as active zone membranes and dense projections. For data to be valid, the analysis must be repeated from hundreds or thousands of images from several biological replicates, a daunting task. Here we report a custom computer program to analyze these features: SynapsEM. In short, we developed macros for ImageJ/Fiji to record x,y-coordinates of segmented structures; the coordinates are then exported as text files. Independent investigators can reload the images and text files to confirm or re-evaluate the segmentation using ImageJ. The Matlab program calculates and reports key synaptic parameters from the coordinates. Since the values are calculated, rather than measured from each micrograph, other parameters can be extracted in Matlab by additional scripting. Thus, this program can accelerate morphometry of synapses and promote a more comprehensive analysis of synaptic ultrastructure.

neuroscience↗

bioRxiv2019

Phospholipase D Transduces Force to TREK-1 Channels in a Biological Membrane

Rapid conversion of force into a biological signal enables living cells to respond to mechanical forces in their environment. The force is believed to initially affect the plasma membrane and then alter the behavior of membrane proteins. Phospholipase D2 (PLD2) is a mechanosensitive enzyme that is regulated by a structured membrane-lipid site comprised of cholesterol and saturated ganglioside (GM1). Here we show stretch activation of TWIK-related K+ channel (TREK-1) is mechanically evoked by PLD2 and spatial patterning involving ordered GM1 and 4,5- bisphosphate (PIP2) clusters. First, mechanical force deforms the ordered lipids, which lowers membrane cholesterol, disrupts the interaction of PLD2 with the GM1 lipids, and allows a complex of TREK-1 and PLD2 to associate with PIP2 clusters. The association with PIP2 activates the enzyme, which produces the second messenger phosphatidic acid (PA) that gates the channel. Co-expression of catalytically inactive PLD2 inhibits TREK-1 stretch currents in a biological membrane. Cellular uptake of cholesterol inhibits TREK-1 currents in culture and depletion of cholesterol from astrocytes releases TREK-1 from GM1 lipids in mouse brain. Depletion of the PLD2 ortholog in flies results in hypersensitivity to mechanical force. We conclude PLD2 mechanosensitivity combines with TREK-1 ion permeability to elicit a mechanically evoked response. SummaryShear thinning activates TREK-1 through a second messenger.

neuroscience↗

bioRxiv2018

Synaptic vesicles undock and then transiently dock after an action potential

Synaptic vesicles fuse with the plasma membrane to release neurotransmitter following an action potential, after which new vesicles must dock to refill vacated release sites. To capture synaptic vesicle exocytosis at cultured mouse hippocampal synapses, we induced single action potentials by electrical field stimulation then subjected neurons to high-pressure freezing to examine their morphology by electron microscopy. During synchronous release, multiple vesicles can fuse at a single active zone; this multivesicular release is augmented by increasing extracellular calcium. Fusions during synchronous release are distributed throughout the active zone, whereas fusions during asynchronous release are biased toward the center of the active zone. Immediately after stimulation, the total number of docked vesicles across all synapses decreases by [~]40%. Between 8 and 14 ms, new vesicles are recruited to the plasma membrane and fully replenish the docked pool in a calcium-dependent manner, but docking of these vesicles is transient and they either undock or fuse within 100 ms. These results demonstrate that recruitment of synaptic vesicles to release sites is rapid and reversible.

neuroscience↗

bioRxiv2016

The NCA-1 ion channel functions downstream of Gq and Rho to regulate locomotion in C. elegans

The heterotrimeric G protein Gq positively regulates neuronal activity and synaptic transmission. Previously, the Rho guanine nucleotide exchange factor Trio was identified as a direct effector of Gq that acts in parallel to the canonical Gq effector phospholipase C. Here we examine how Trio and Rho act to stimulate neuronal activity downstream of Gq in the nematode Caenorhabditis elegans. Through two forward genetic screens, we identify the cation channels NCA-1 and NCA-2, orthologs of mammalian NALCN, as downstream targets of the Gq/Rho pathway. By performing genetic epistasis analysis using dominant activating mutations and recessive loss-of-function mutations in the members of this pathway, we show that NCA-1 and NCA-2 act downstream of Gq in a linear pathway. Through cell-specific rescue experiments, we show that function of these channels in head acetylcholine neurons is sufficient for normal locomotion in C. elegans. Our results suggest that NCA-1 and NCA-2 are physiologically relevant targets of neuronal Gq-Rho signaling in C. elegans.

genetics↗