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SPIN90 modulates the architecture of lamellipodial actin in an ARPC5L dependent fashion.

Luyan CaoiD, Angika BasantiD, Miroslav MladenoviD, Naoko KogataiD, Antoine G. JegouiD, Guillaume Romet‐LemonneiD, Sophie BrasseletiD, Manos MavrakisiD, Michael WayiD

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Abstract

The Arp2/3 complex in mammals, consists of 8 iso-complexes with different properties as there are two isoforms of the Arp3, ArpC1 and ArpC5 subunits. All iso-complexes still generate actin branches when activated by class 1 nucleation promotion factors. Using recombinant Arp2/3 iso-complexes with defined compositions, we now demonstrate that SPIN90 selectively activates ArpC5L- rather than ArpC5- containing complexes to generate linear actin filaments. Consistent with this, SPIN90 at the leading edge of migrating cells enhances the recruitment of ArpC5L-, but not ArpC5- containing, Arp2/3 complexes to lamellipodia. These SPIN90-Arp2/3-ArpC5L complexes generate linear actin filaments that integrate into lamellipodia and impact protrusion efficiency. Moreover, using polarized light microscopy, we show that loss of SPIN90 leads to changes in the lamellipodial actin filament architecture. Our results demonstrate that SPIN90 regulates the organisation and dynamics of lamellipodial actin in an Arp2/3 iso-complex dependent fashion.

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Claude Opus 5.5 · xhigh effort · 7 Nov 2026 · from the full text of version 3 (3 Oct 2026) · CC BY 4.0

SPIN90 selectively activates ArpC5L-containing Arp2/3 complexes to make linear actin filaments, shaping lamellipodial filament orientation and protrusion efficiency in B16 melanoma cells.

It was unclear whether every Arp2/3 iso-complex can make linear filaments when activated by SPIN90, because the earlier SPIN90-Arp2/3 structure used a natural bovine complex without isoform-level resolution. Here defined recombinant iso-complexes, knockout cells and polarized fluorescence microscopy tie the ArpC5L preference to lamellipodial actin architecture.

Key results

  1. ShownSPIN90 efficiently activates only ArpC5L-containing Arp2/3 complexes to nucleate linear filaments, whichever ArpC1 isoform they carry. ArpC5-containing complexes are barely activated.

    TIRF microscopy: ArpC5L complexes produced linear filaments with 20- to 40-fold greater efficiency with SPIN90. Pyrene assays showed ArpC5 complexes only slightly above controls. · Spin90 preferentially activates…

  2. ShownSPIN90 binds ArpC5L-containing complexes more strongly than ArpC5-containing ones, which probably explains the activation difference.

    GST pull-down with ArpC1B/ArpC5L: KD 3.01 ± 0.85 μM, Hill coefficient 1.59 ± 0.67. ArpC5 complex pull-down was too weak to fit a KD. Dissociation rates were similar (0.067 min^-1 for ArpC5, 0.045 min^-1 for ArpC5L). · SPIN90 has a higher affinity for Arp2/3…

  3. ShownIn B16 cells SPIN90 is enriched at the leading edge and flows rearward with ArpC5L. Depleting SPIN90 lowers leading-edge ArpC5L but does not lower ArpC5.

    ArpC5L protein is ~1.7-fold higher than ArpC5 in B16 cells. SPIN90 knockdown reduced NeonGreen-ArpC5L at the leading edge, also when ArpC5 was depleted. The NeonGreen-ArpC5 signal increased. SPIN90 Nter, which cannot bind Arp2/3, did not flow rearward. · Spin90 preferentially activates…; SPIN90 is enriched at the leading edge; Both SPIN90 and Arp2/3 integrate into…; SPIN90 enhances recruitment of ArpC5L but not…

  4. ShownLoss of SPIN90 or ArpC5L narrows the distribution of lamellipodial actin filament orientations toward perpendicular to the leading edge. ArpC5 loss broadens it. Overexpressing full-length SPIN90 widens it.

    Polarized fluorescence (PyPOLAR): angular spread ψ was high (mean 157°) in WT and all KO lines with no detectable difference. Mean-orientation distributions were broader in ArpC5 KO and narrower in ArpC5L KO and SPIN90 KO than in WT. Ruby-SPIN90 gave a wider distribution than Ruby-SPIN90 Nter. · SPIN90 regulates lamellipodial actin…

  5. ShownSPIN90 loss lowers lamellipodial protrusion efficiency and migration speed, like ArpC5 loss, while ArpC5L KO leaves them near WT. SPIN90 overexpression raises the protrusion rate.

    FRAP of GFP-β-actin: SPIN90 KO had a slower protrusion rate and reduced protrusion efficiency (protrusion rate divided by actin polymerisation rate). ArpC5 KO and SPIN90 KO cells migrated more slowly than WT and ArpC5L KO in random migration. Lamellipodial actin density rose in SPIN90 KO and ArpC5L KO and fell in ArpC5 KO. · SPIN90 regulates lamellipodial protrusion

Methods, data and reproducibility

Recombinant human Arp2/3 iso-complexes were tested with SPIN90 in pyrene-actin, TIRF and GST pull-down assays. In mouse B16-F1 cells, CRISPR knockouts, siRNA, endogenous NeonGreen and GFP knock-ins, live imaging and polarized fluorescence microscopy of phalloidin-stained actin were used.

  • Recombinant human Arp2/3 iso-complexes (ArpC1A/B with ArpC5/ArpC5L, Arp3 background) with human SPIN90 C-terminus (267-715), rabbit muscle actin · human (recombinant proteins); rabbit muscle actin · n = each experiment repeated three times unless stated · UniProt Q9NZQ3-3 (SPIN90 267-715); complexes P61160, P61158, Q92747, O15143, O15144, O15145, P59998, Q9H9F9
  • B16-F1 melanoma cells: ArpC5, ArpC5L and SPIN90 CRISPR KO lines, endogenous NeonGreen-ArpC5 or ArpC5L-NeonGreen and GFP-β-actin knock-ins, siRNA depletion, Ruby-SPIN90 expression · mouse · n = >8 cells per condition for live imaging; others in legends · UniProt Q9ESJ4 (mouse SPIN90)
  • Polarized fluorescence microscopy of Alexa-488 phalloidin-stained lamellipodia (18 polarization angles, 10 pixels from the leading edge) in WT, KO and Ruby-SPIN90 cells · mouse
Methods & tools
Pyrene actin polymerisation assay, TIRF microscopy, GST pull-down, PyPOLAR, FIJI (GelAnalyzer, Multi Kymograph), CRISPR-Cas9 / ORANGE knock-in, VT-iSIM, Livecyte, PyMOL, Prism 10
Reagents & environment
Prism 10 (version 10.1.1); MetaMorph 7.8.13.0 for fixed-cell imaging; PyPOLAR (RRID:SCR_024681) used for polarized microscopy analysis.
Reproducibility gaps
No code or data repository URLs are given; sample sizes and p-values are deferred to figure legends, which are not in the text.
Limitations (4)
  • SPR, microscale thermophoresis and ITC gave no reproducible results, so affinity rests on GST pull-downs. No KD could be fitted for ArpC5-containing complexes because too little protein was pulled down. · stated by the authors
  • Polarized fluorescence microscopy gives mean orientation and alignment at about 200 nm scale but not the orientation of individual filaments, which EM provides. · stated by the authors
  • How actin architecture and dynamics together determine protrusion efficiency, especially under different external mechanical conditions, remains to be established. · stated by the authors
  • Antibodies did not work for endogenous SPIN90 localisation, so localisation relies on overexpressed Ruby-SPIN90. Linear filaments were not directly visualised in cells; their role is inferred from orientation distributions and KO phenotypes in a single cell line (B16). · noted in this summary

Who should read:Read this if you study Arp2/3 isoform specificity, SPIN90-driven linear actin nucleation, or use polarized fluorescence to quantify actin filament orientation in lamellipodia.

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