The cellular processes underpinning life are orchestrated by proteins and their interactions. The associated structural and dynamic heterogeneity, despite being key to function, poses a fundamental challenge to existing analytical and structural methodologies. We used interferometric scattering microscopy to quantify the mass of single biomolecules in solution with 2% sequence mass accuracy, up to 19-kilodalton resolution, and 1-kilodalton precision. We resolved oligomeric distributions at high dynamic range, detected small-molecule binding, and mass-imaged proteins with associated lipids and sugars. These capabilities enabled us to characterize the molecular dynamics of processes as diverse as glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomolecular interactions, one molecule at a time.

Tropomyosin isoforms play an important role in the organisation of cytoplasmic actomyosin complexes in regard to function and cellular localisation. In particular, Tpm4.2 is upregulated in rapidly migrating cells and responsible for the specific recruitment of the cytoplasmic class-2 myosin NM-2A to actin filaments during the formation of stress fibres. Here, we investigate how the decoration of F-actin with Tpm4.2 affects the motor properties of NM-2A under conditions of low and high load. In the absence of external forces, decoration of actin filaments with Tpm4.2 does not affect the gated release of ADP from NM-2A and the transition from strong to weak actin-binding states. In the presence of resisting loads, our results reveal a marked increase in the mechanosensitive gating between the leading and trailing myosin head. Thereby, the processive behaviour of NM-2A is enhanced in the presence of resisting loads. The load- and Tpm4.2-induced changes in the functional behaviour of NM-2A are in good agreement with the role of this myosin in the context of stress fibres and the maintenance of cellular tension.

The metazoan actin cytoskeleton supports a wide range of contractile and transport processes. Recent studies have shown how the dynamic association with specific tropomyosin isoforms generates actin filament populations with distinct functional properties. However, critical details of the associated molecular interactions remain unclear. Here, we report the properties of actomyosin–tropomyosin complexes containing filamentous β-actin, nonmuscle myosin-2B (NM-2B) constructs, and either tropomyosin isoform Tpm1.8cy (b.–.b.d), Tpm1.12br (b.–.b.c), or Tpm3.1cy (b.–.a.d). Our results show the extent to which the association of filamentous β-actin with these different tropomyosin cofilaments affects the actin-mediated activation of NM-2B and the release of the ATP hydrolysis products ADP and phosphate from the active site. Phosphate release gates a transition from weak to strong F-actin–binding states. The release of ADP has the opposite effect. These changes in dominant rate-limiting steps have a direct effect on the duty ratio, the fraction of time that NM-2B spends in strongly F-actin–bound states during ATP turnover. The duty ratio is increased ∼3-fold in the presence of Tpm1.12 and 5-fold for both Tpm1.8 and Tpm3.1. The presence of Tpm1.12 extends the time required per ATP hydrolysis cycle 3.7-fold, whereas it is shortened by 27 and 63% in the presence of Tpm1.8 and Tpm3.1, respectively. The resulting Tpm isoform–specific changes in the frequency, duration, and efficiency of actomyosin interactions establish a molecular basis for the ability of these complexes to support cellular processes with widely divergent demands in regard to force production, capacity to move processively, and speed of movement.

In spite of their great importance in biology, methods providing access to spontaneous molecular interactions with and on biological membranes have been sparse. The recent advent of mass photometry to quantify mass distributions of unlabeled biomolecules landing on surfaces raised hopes that this approach could be transferred to membranes. Here, by introducing a new interferometric scattering (iSCAT) image processing and analysis strategy adapted to diffusing particles, we enable mass-sensitive particle tracking (MSPT) of single unlabeled biomolecules on a supported lipid bilayer. We applied this approach to the highly nonlinear reaction cycles underlying MinDE protein self-organization. MSPT allowed us to determine the stoichiometry and turnover of individual membrane-bound MinD/MinDE protein complexes and to quantify their size-dependent diffusion. This study demonstrates the potential of MSPT to enhance our quantitative understanding of membrane-associated biological systems.

Cytoplasmic b-actin supports fundamental cellular processes in healthy and diseased cells including cell adhesion, migration, cytokinesis and maintenance of cell polarity. Mutations in ACTB, the gene encoding cytoplasmic b-actin, lead to severe disorders with a broad range of symptoms. The two dominant heterozygous gain-of-function b-actin mutations p.R183W and p.E364K were identified in patients with developmental malformations, deafness and juvenile-onset dystonia (p.R183W) and neutrophil dysfunction (p.E364K). Here, we report the recombinant production and functional characterization of the two mutant proteins. Arg183 is located near the nucleotide-binding pocket of actin. Our results from biochemical studies and molecular dynamics simulations show that replacement by a tryptophan residue at position 183 establishes an unusual stacking interaction with Tyr69 that perturbs nucleotide release from actin monomers and polymerization behavior by inducing a closed state conformation. The replacement of Glu364 by a lysine residue appears to act as an allosteric trigger event leading to the preferred formation of the closed state. Thus, our approach indicates that both mutations affect interdomain mobility and nucleotide interactions as a basis for the formation of disease phenotypes in patients. IntroductionActin is a highly conserved and ubiquitous protein found in nearly all eukaryotic cells. Six actin isoforms can be distinguished in vertebrates: three a-actin isoforms (a-skeletal muscle, a-cardiac muscle and a-aortic smooth muscle, also known as a-vascular), one b-isoform (b-cytoplasmic) and two c-isoforms (c-cytoplasmic and c-smooth muscle also known as centeric). Cytoplasmic b-and c-actin are essential for cell migration, cell shape maintenance, mitosis and intracellular transport processes and are expressed at moderate to high levels in nearly all adult tissues [1,2]. The most abundant isoactin in many nonmuscle cells including myeloid and neuronal cells is b-actin [2]. Cellular studies show that the b-isoform is preferentially recruited into cellular protrusions, stress fibers, circular bundles and at cell-cell contacts [3,4]. The rapid cytoskeletal rearrangements observed for these structures appear to be linked to the highly dynamic turnover of actin filaments made from b-actin [5].All actin isoforms share the same architecture with four different subdomains and a common nucleotidebinding site. In vivo, b-actin is post-translationally modified by cleavage of the first methionine followed by N-terminal acetylation. Actin is an ATPase and the hydrophobic nucleotide-binding site is located in the cleft between subdomains SD1-2 and SD3-4 (Fig. 1A). In the presence of divalent cations, monomers with bound ATP assemble into filaments. The growth of actin filaments depends on the addition of actin-ATP monomers predominantly at the fast-growing barbed ends. After monomer addition, the bound ATP is hydrolyzed to ADP and inorganic phosphate (P i ) followed by P i -release. Because dissociation of actin-ADP occurs pr...