Biology subjects

Clark, M. G.

Publications and source records attributed to Clark, M. G..

4 recordsLinked to original sources

bioRxiv2025

Real-Time and Site-Specific Perturbation of Dynamic Subcellular Compartments Using Femtosecond Pulses

Understanding laser interactions with subcellular compartments is crucial for advancing optical microscopy, phototherapy, and optogenetics. While continuous-wave (CW) lasers rely on linear absorption, femtosecond (fs) lasers enable nonlinear multiphoton absorption confined to the laser focus, offering high axial precision. However, current fs laser delivery methods lack the ability to target dynamic molecular entities and automate target selection, limiting real-time perturbation of biomolecules with mobility or complex distribution. Additionally, existing technologies separate fs pulse delivery and imaging, preventing simultaneous recording of cellular responses. To overcome these challenges, we introduce fs real-time precision opto-control (fs-RPOC), which integrates a laser scanning microscope with a closed-loop feedback mechanism for automated, chemically selective subcellular perturbation. fs-RPOC achieves superior spatial precision and fast response time, enabling single- and sub-organelle microsurgery of dynamic targets and localized molecular modulation. By applying a pulse-picking method, fs-RPOC independently controls laser average and peak power at any desired subcellular compartment. Targeting mitochondria, fs-RPOC reveals site-specific molecular responses resulting from fs-laser-induced ROS formation, H2O2 diffusion, and low-density plasma generation. These findings offer new insights into fs laser interactions with subcellular compartments and demonstrate fs-RPOCs potential for precise molecular and organelle regulation.

bioengineering↗

bioRxiv2023

Spatiotemporal precise optical manipulation of intracellular molecular activities

Controlling chemical processes in live cells is a challenging task. The spatial heterogeneity of biochemical reactions in cells is often overlooked by conventional means of incubating cells with desired chemicals. A comprehensive understanding of spatially diverse biochemical processes requires precise control over molecular activities at the subcellular level. Herein, we develop a closed-loop optoelectronic control system that allows the manipulation of biomolecular activities in live cells at high spatiotemporal precision. Chemical-selective fluorescence signals are utilized to command lasers that trigger specific chemical reactions or control the activation of photoswitchable inhibitors at desired targets. We demonstrate the capability to selectively produce reactive oxygen species (ROS) solely at targeted organelles using blue light. Notably, the induction of ROS in the endoplasmic reticulum leads to a more pronounced disruption of tubulin polymerization and a reduction in green fluorescent protein signals, in comparison to that in lipid droplets. Moreover, when combined with a photoswitchable inhibitor, we selectively inhibit tubulin polymerization within subcellular compartments. This technology enables spatiotemporal control over chemical processes and drug activities, exclusively at desired targets, while minimizing undesired effects on non-targeted locations.

cell biology↗

bioRxiv2023

Amyloid β Induces Lipid Droplet-Mediated Microglial Dysfunction in Alzheimer's Disease

Several microglia-expressed genes have emerged as top risk variants for Alzheimers disease (AD). Impaired microglial phagocytosis is one of the main proposed outcomes by which these AD-risk genes may contribute to neurodegeneration, but the mechanisms translating genetic association to cellular dysfunction remain unknown. Here we show that microglia form lipid droplets (LDs) upon exposure to amyloid-beta (A{beta}), and that their LD load increases with proximity to amyloid plaques in brains from human patients and the AD mouse model 5xFAD. LD formation is dependent on age and disease progression and is prominent in the hippocampus in mice and humans. Despite differences in microglial LD load between brain regions and sexes in mice, LD-laden microglia exhibited a deficit in A{beta} phagocytosis. Unbiased lipidomic analysis identified a decrease in free fatty acids (FFAs) and a parallel increase in triacylglycerols (TGs) as the key metabolic transition underlying LD formation. DGAT2, a key enzyme for converting FFAs to TGs, promotes microglial LD formation and is increased in 5xFAD and human AD brains. Inhibition or degradation of DGAT2 improved microglial uptake of A{beta} and drastically reduced plaque load in 5xFAD mice, respectively. These findings identify a new lipid-mediated mechanism underlying microglial dysfunction that could become a novel therapeutic target for AD.

neuroscience↗

bioRxiv2022

Real-time precision opto-control of chemical processes in live cells

Precision control of molecular activities and chemical reactions in live cells is a long-sought capability by life scientists. No existing technology can probe molecular targets in cells and simultaneously control the activities of only these targets at high spatial precision and on the fly. We develop a real-time precision opto-control (RPOC) technology that detects a chemical-specific optical response from molecular targets during laser scanning and uses the optical signal to trigger an acousto-optic modulator, which allows a separate laser beam to only interact with the molecules of interest without affecting other parts of the sample. RPOC can automatically probe and control biomolecular activities and chemical processes in dynamic living samples with submicron spatial accuracy, nanoseconds response time, and high chemical specificity.

bioengineering↗