CYRIA

CYRI-A is a RAC1-interacting protein that negatively regulates actin dynamics to control macropinocytosis and integrin trafficking (PMID:34165494, PMID:33217330). It is transiently recruited to nascent macropinosomes in a PI3K- and RAC1-dependent manner, arriving after RAC1 and actin signaling but before RAB5A, consistent with a role as a local inhibitor of actin polymerization at these sites (PMID:34165494). Mechanistically, CYRI-family proteins bind active GTP-bound RAC1 through an N-terminal RAC1-binding subdomain and compete with the Scar/WAVE complex for this interaction, establishing a negative feedback loop on RAC1-driven actin assembly (PMID:33217330). CYRI-A and its paralog CYRI-B can additionally form autoinhibited homo- and heterodimers, adding a further regulatory layer to RAC1 signaling (PMID:33217330). Functionally, loss of CYRI proteins reduces internalization of high-molecular-weight dextran, confirming their requirement for macropinocytosis (PMID:35530513), while co-depletion of CYRI-A and CYRI-B elevates surface α5β1 integrin via reduced internalization and enhances cell migration, invasion, and anchorage-independent growth (PMID:34165494).

1. 2020 High

   Establishing how CYRI proteins act on RAC1 was needed to explain their effect on actin; the structure showed CYRI-B binds active RAC1 and competes with Scar/WAVE, defining a negative-feedback mechanism on actin dynamics.

   Evidence Crystal structure of CYRI-B:RAC1Q61L complex with competitive binding assays

   PMID:33217330

   Open questions at the time

   • CYRI-A itself was characterized largely as a family member by dimerization behavior, not by an independent CYRI-A:RAC1 structure
   • the cellular consequence of dimerization on RAC1 binding was not functionally dissected
   • stoichiometry and dynamics of CYRI vs Scar/WAVE competition in cells not resolved
2. 2020 High

   Beyond a single inhibitory mode, it was unknown how CYRI activity is itself regulated; demonstrating that CYRI-A and CYRI-B form autoinhibited homo- and heterodimers added an additional regulatory layer to RAC1 signaling.

   Evidence Biochemical analysis of CYRI-A/B dimerization alongside crystallographic domain architecture analysis

   PMID:33217330

   Open questions at the time

   • trigger and kinetics of dimer assembly/disassembly in cells unknown
   • relative abundance and biological role of homodimers vs heterodimers not defined
3. 2021 High

   Where and when CYRI-A acts in the cell was unknown; live imaging placed it transiently at nascent macropinosomes downstream of PI3K/RAC1/actin and before RAB5A, localizing its inhibitory function to an early macropinosome stage.

   Evidence Live-cell imaging, subcellular localization, PI3K/RAC1 pharmacological inhibition, and depletion in cultured cells

   PMID:34165494

   Open questions at the time

   • molecular signal driving the transient recruitment and departure not identified
   • whether RAB5A timing is a direct consequence of CYRI-A action untested
4. 2021 Medium

   The downstream physiological output of CYRI loss was unclear; co-depletion of CYRI-A and CYRI-B raised surface α5β1 integrin via reduced internalization and increased migration, invasion, and 3D growth, linking CYRI to integrin trafficking and invasive behavior.

   Evidence siRNA knockdown, surface integrin flow cytometry, Transwell migration/invasion and 3D growth assays

   PMID:34165494

   Open questions at the time

   • CYRI-A-specific contribution not separated from CYRI-B in the co-depletion phenotype
   • direct mechanistic link between actin/macropinosome regulation and integrin internalization not established
5. 2022 Medium

   A dedicated functional readout was needed to confirm the macropinocytosis role; CYRI-depleted cells internalized less high-molecular-weight dextran, directly confirming a requirement for CYRI proteins in macropinocytosis.

   Evidence Image-based dextran uptake assay with fluorescence quantification in CYRI-depleted cells

   PMID:35530513

   Open questions at the time

   • single-method assay from one lab
   • does not separate CYRI-A from CYRI-B contributions to dextran uptake

• It remains unknown how CYRI-A and CYRI-B activities are individually apportioned across macropinocytosis, integrin trafficking, and migration, and how dimerization-based autoinhibition is dynamically controlled in living cells.
• no CYRI-A-specific loss-of-function dissection separating it from CYRI-B
• no characterization of the upstream signal that toggles dimer autoinhibition