Design on Solid Ground

 Quick Analysis Plan Copied

 Long Analysis Plan

Rationale from existing evidence

The fla10 kinesin-II temperature-sensitive allele is the standard cheap probe for anterograde IFT dependence: FAP163 flagellar levels drop in fla10 at restrictive temperature, confirming IFT trafficking for that protein FAP163 fla10 shift result. HSP70A is already known to be present in the flagellar matrix (~10% of cellular pool), punctate along the flagellum, concentrated at tips, and >90% colocalized with FLA10 HSP70A flagellar matrix and FLA10 colocalization — exactly the pattern expected of an IFT passenger, but colocalization alone cannot exclude diffusion. The temperature shift is the missing discriminator.

Experimental design

• Strains: fla10 ts mutant (e.g., CC-2637 background) plus isogenic WT; both carrying endogenous or HA-tagged HSP70A (tagged CC-2929-style transformant as secondary readout).
• Protocol: Grow at 21°C with full-length flagella; shift to 32°C (restrictive). Harvest flagella at 0, 15, 30, 60, 120 min by pH-shock or dibucaine deflagellation.
• Readouts: (1) Anti-HSP70A immunoblot of flagellar matrix normalized to axonemal tubulin and total cellular HSP70A; (2) anti-HSP70A/anti-FLA10 double immunofluorescence for tip-enrichment loss; (3) FAP163 or kinesin-II itself as positive IFT-trafficking control; (4) IFT46 depletion as blockage confirmation.
• Interpretation: Flagellar HSP70A halving within ~60–120 min (mirroring FAP163) → IFT-dependent import (cargo hypothesis). Persistence at high levels → passive diffusion through the transition zone dominates. Partial/slow loss → mixed mechanism.
• Critical controls: permissive-temperature time course (rules out photobleaching/turnover artifacts), WT at 32°C (rules out heat-shock-driven HSP70A upregulation masking loss — note 40°C heat shock strongly induces HSP70A transcription in Chlamydomonas HSP70A heat inducibility control consideration, and 32°C induction must be checked), and flagellar length verification (IFT blockage shortens flagella).

Predicted outcomes (simulated hypothesis space, not observed data)

Simulated curves only, assuming first-order export/turnover for the cargo case (~50% loss/hour, ±10% shading reflects measurement uncertainty), permissive turnover for the diffusion case, and a two-compartment mix. These are hypothetical expectations to pre-register, not measured data. Note the confound: heat-inducible HSP70A synthesis at 32°C could offset flagellar loss in the cargo case; cycloheximide addition or transcriptional monitoring (RT-qPCR) is required to keep the readout interpretable HSF1-driven HSP70A induction confounder.

Known unknowns

• No published data in the supplied records quantify flagellar HSP70A turnover rate or half-life — the decay kinetics above are assumptions, not evidence.
• The 32°C shift may partially (not fully) inactivate kinesin-II; a dose–response across 25/27/29/32°C would bound the residual-transport artifact.
• Colocalization >90% does not establish physical binding; no direct HSP70A–IFT interaction was biochemically resolved in the original study (cofractionation failed to identify interactors), so a negative result would still need an orthogonal IFT-independent-entry test (e.g., transition-zone permeability measurement with size-excluded dextrans).