optogenetic membrane potential perturbation

Stages

1. 1.
   Kir perturbation across neutrophil chemotaxis models(broad_screen)

   This stage tests whether inwardly rectifying potassium channels are required for directional sensing in neutrophils across multiple models.

   Selection: disruption of directional sensing toward different chemoattractants after Kir blocking or knockout

2. 2.
   Voltage imaging during zebrafish neutrophil chemotaxis(functional_characterization)

   This stage characterizes how membrane potential changes during neutrophil migration and whether Kir7.1 is required for depolarization toward the chemokine source.

3. 3.
   Optogenetic causal perturbation of membrane potential(confirmatory_validation)

   This stage tests whether imposed membrane-potential changes can directly bias pseudopod selection, trigger protrusions, or stall migration.

   Selection: behavioral response to focal depolarization or global hyperpolarization

4. 4.
   GPCR signaling analysis in dHL-60 cells(secondary_characterization)

   This stage extends the migration findings to a signaling readout in a neutrophil-like cell model.

Steps

1. 1.
   Block or knock out inwardly rectifying potassium channels in neutrophils

   Test whether Kir activity is necessary for directional sensing during chemotaxis.

   Necessity testing provides an initial functional readout before deeper mechanistic characterization.

2. 2.
   Image membrane potential dynamics with genetically encoded voltage indicators in zebrafish neutrophilsassay readout

   Measure endogenous membrane-potential changes during chemotaxis and relate them to cell behavior.

   After establishing that Kir perturbation affects directional sensing, voltage imaging reveals the dynamic membrane-potential states associated with migration.

3. 3.
   Apply focal optogenetic depolarization to bias protrusion selectioncausal perturbation method

   Test whether local depolarization is sufficient to bias pseudopod selection and trigger new protrusions.

   This follows observational voltage imaging to move from correlation to causal sufficiency testing.

4. 4.
   Apply global hyperpolarization to test effects on migration progressioncausal perturbation method

   Test whether whole-cell hyperpolarization inhibits ongoing migration.

   A global perturbation complements focal depolarization by testing whether broad voltage shifts suppress motility rather than redirect it.

5. 5.
   Assess GPCR signaling activation in dHL-60 cells under Kir-related conditions

   Link Kir function to GPCR signaling activation in a neutrophil-like cell model.

   This step provides signaling-level mechanistic support after behavioral and voltage-perturbation observations.