Edelstein, S. J. | Search bioRxiv

bioRxiv2022

RevGel-seq: instrument-free single-cell RNA sequencing using a reversible hydrogel for cell-specific barcoding

Progress in sample preparation for scRNA-seq is reported based on RevGel-seq, a reversible-hydrogel technology optimized for samples of fresh cells. Complexes of one cell paired with one barcoded bead are stabilized by a chemical linker and dispersed in a hydrogel in the liquid state. Upon gelation on ice the complexes are immobilized and physically separated without requiring nanowells or droplets. Cell lysis is triggered by detergent diffusion, and RNA molecules are captured on the adjacent barcoded beads for further processing with reverse transcription and preparation for cDNA sequencing. As a proof of concept, analysis of PBMC using RevGel-seq achieves results similar to microfluidic-based technologies when using the same original sample and the same data analysis software. In addition, a clinically relevant application of RevGel-seq is presented for pancreatic islet cells. Furthermore, characterizations carried out on cardiomyocytes demonstrate that the hydrogel technology readily accommodates very large cells. Standard analyses are in the 10,000-input cell range with the current gelation device, in order to satisfy common requirements for single-cell research. A convenient stopping point after two hours has been established by freezing at the cell lysis step, with full preservation of gene expression profiles. Overall, our results show that RevGel-seq represents an accessible and efficient instrument-free alternative, enabling flexibility in terms of experimental design and timing of sample processing, while providing broad coverage of cell types.

cell biology↗

bioRxiv2019

Neurogranin Stimulates Ca2+/calmodulin-dependent Kinase II by Inhibiting Calcineurin at Specific Calcium Spike Frequencies

Calmodulin sits at the centre of molecular mechanisms underlying learning and memory. Its complex, and sometimes opposite influences, via the binding to various proteins, are yet to be fully understood. Calcium/calmodulin-dependent protein kinase II (CaMKII) and calcineurin (CaN) both bind open calmodulin, favouring Long-term potentiation (LTP) or depression (LTD) respectively. Neurogranin binds to the closed conformation of calmodulin and its impact on synaptic plasticity is less clear. We set up a mechanistic computational model based on allosteric principles to simulate calmodulin state transitions and its interaction with calcium ions and the three binding partners mentioned above. We simulated calcium spikes at various frequencies and show that neurogranin regulates synaptic plasticity along three modalities. At low spike frequencies, neurogranin inhibits the onset of LTD by limiting CaN activation. At intermediate frequencies, neurogranin limits LTP by precluding binding of CaMKII with calmodulin. Finally, at high spike frequencies, neurogranin promotes LTP by enhancing CaMKII autophosphorylation. While neurogranin might act as a calmodulin buffer, it does not significantly preclude the calmodulin opening by calcium. On the contrary, neurogranin synchronizes the opening of calmodulins two lobes and promotes their activation at specific frequencies, increasing the chance of CaMKII trans-autophosphorylation. Taken together, our study reveals dynamic regulatory roles played by neurogranin on synaptic plasticity, which provide mechanistic explanations to opposing experimental findings.\n\nAuthor SummaryHow our brains learn and remember things lies in the subtle changes of the strength with which brain cells connect to each other, the so-called synaptic plasticity. At the level of the recipient neuron, some of the information is encoded into patterns of intracellular calcium spikes. Calmodulin, a small bilobed protein which conformation is regulated by the binding of calcium ions, decodes these signals, and modulates the activity of specific binding partners.\n\nTwo key regulators, calcineurin and calcium/calmodulin-dependent protein kinase II, which respectively weaken or strengthen synaptic connections, bind both lobes of calmodulin in its open form, favoured by calcium. On the contrary, neurogranin binds preferentially to one lobe of calmodulin, in the closed form, unfavoured by calcium. It was thus initially suggested that it would inhibit calmodulin activation and decrease synaptic plasticity. However, past research showed that neurogranin sometimes actually enhances synaptic plasticity, though the mechanism was unclear.\n\nOur computational models showed that neurogranin synchronizes the activation of the two lobes of calmodulin, favouring opening at high frequency calcium spikes. By doing so, neurogranin increases the impact of calmodulin on calcium/calmodulin-dependent protein kinase II and reduces its effect on calcineurin, resulting in a strengthening of synaptic connections.

neuroscience↗