Single-Cell Mechanobiology

BioPen in an ideal tool to study the kinetics biochemical processes on a single-cell level giving you full control of the entire experimental procedure using a confined flow that limits exposure to your region of interest.

With its capability to deliver what you want, where and when you want it, you can create localized chemical environments in open solution to study processes such as chemotaxis on a single-cell level. Create chemical gradients across cells or tissues to study the effect on cytoskeleton and motor protein organization.

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In the paper A Genetically Encoded Biosensor Strategy for Quantifying Non-muscle Myosin II Phosphorylation Dynamics in Living Cells and Organisms by M.A. Rizzo and coworkers, Regulatory Light Chain (RLC) phosphorylation was investigated during retraction of cell protrusion using a Förster Resonance Energy Transfer (FRET) approach. To induce the retraction, the cell protrusion was stimulated with local delivery of a Platelet-Derived Growth Factor (PDGF) using the BioPen PRIME system.

PDGF stimulation resulted in the retraction of a cell protrusion initiating 5 min into the treatment. The morphology of the cell outside of the treatment area did not undergo substantial alterations. Before retraction, the tip of the protrusion contained stable high-anisotropy phosphorylated regions. Retraction proceeded through a multi-phasic process similar to those during random migration. Initially, cytoskeletal movements were observed 10–20 μm deep into the protrusion .

Single Muscle Fiber Physiology

Using the BioPen (formerly the Multifunctional pipette), compounds were delivered locally to the end or side of single adult mouse skeletal muscle fibres to test whether changes in mitochondrial membrane potential were transmitted to more distant located mitochondria.

Mitochondrial membrane potential was monitored with tetramethylrhodamine ethyl ester (TMRE). Cytosolic free [Ca2+] was monitored with fluo-3. A pulse of carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) applied to a small area of the muscle fibre (30 µm in diameter) which produced a rapid decrease in the mitochondrial TMRE signal (indicative of depolarization) to 38% of its initial value. After washout of FCCP, the TMRE signal partially recovered.

At distances greater than 50 µm away from the site of FCCP application, the mitochondrial TMRE signal was unchanged. Similar results were observed when two sites along the fibre were pulsed sequentially with FCCP. After a pulse of FCCP, cytosolic [Ca2+] was unchanged and fibres contracted in response to electrical stimulation.

These results indicate that extensive networks of interconnected mitochondria do not exist in skeletal muscle. Furthermore, the limited and reversible effects of targeted FCCP application with the BioPen highlight its advantages over bulk application of compounds to isolated cells.

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