biostudies-literatureBleicher PIntramural NIH HHSe07772https://www.ebi.ac.uk/biostudies/studies/S-EPMC12802541biostudies-literatureUnknown22(3)Cells express three non-muscle myosin 2 (NM2) paralogs that form bipolar filaments of ≈30 motors each. Of these, NM2A and NM2B are best studied and show distinct enzymatic and mechanical properties. Although they can colocalize in cells, they also have unique localization patterns, suggesting functional differences. Most studies have examined these proteins interacting with actin under very low loads, but in cells they likely contribute to generating and maintaining cytoskeletal tension. Inspired by sarcomere-like tension generation in non-muscle cells, a minimal platform is reconstituted to study NM2 activity under load. Using micropatterned formin to align actin filaments in anti-parallel bundles, phosphorylated NM2 filaments are introduced to observe their interactions via TIRF (Total ISmall (Weinheim an der Bergstrasse, Germany)Effect of Load on Non-Muscle Myosin 2 Paralog Filaments in a Biomimetic Contractile Actin Array.PMC12802541Z01 HL001786Combs CASellers JRBillington NHart RQazi SBleicher PChandrasekar IKnutson JRHan DfalseEffect of Load on Non-Muscle Myosin 2 Paralog Filaments in a Biomimetic Contractile Actin Array.Cells express three non-muscle myosin 2 (NM2) paralogs that form bipolar filaments of ≈30 motors each. Of these, NM2A and NM2B are best studied and show distinct enzymatic and mechanical properties. Although they can colocalize in cells, they also have unique localization patterns, suggesting functional differences. Most studies have examined these proteins interacting with actin under very low loads, but in cells they likely contribute to generating and maintaining cytoskeletal tension. Inspired by sarcomere-like tension generation in non-muscle cells, a minimal platform is reconstituted to study NM2 activity under load. Using micropatterned formin to align actin filaments in anti-parallel bundles, phosphorylated NM2 filaments are introduced to observe their interactions via TIRF (Total I2026-01-01T00:00:00Z2026 Jan2026-06-11T04:57:15.935Z2026-06-11T03:08:07.948ZS-EPMC128025414130594310.1002/smll.202507772