Excessive activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome is involved in many chronic inflammatory diseases, including cardiovascular and Alzheimer’s disease. Here we show that microtubule-affinity regulating kinase 4 (MARK4) binds to NLRP3 and drives it to the microtubule-organizing centre, enabling the formation of one large inflammasome speck complex within a single cell. MARK4 knockdown or knockout, or disruption of MARK4-NLRP3 interaction, impairs NLRP3 spatial arrangement and limits inflammasome activation. Our results demonstrate how an evolutionarily conserved protein involved in the regulation of microtubule dynamics orchestrates NLRP3 inflammasome activation by controlling its transport to optimal activation sites, and identify a targetable function for MARK4 in the control of innate immunity.

Subcellular localization of mRNAs by cytoskeletal motors plays critical roles in the spatial control of protein function1. However, optical limitations of studying mRNA transport in vivo mean that there is little mechanistic insight into how transcripts are packaged and linked to motors, and how the movement of mRNA:motor complexes on the cytoskeleton is orchestrated. Here, we have reconstituted transport of mRNPs containing specific RNAs in vitro. We show directly that mRNAs that are either apically localized or non-localized in Drosophila embryos associate with the dynein motor and move bidirectionally on individual microtubules, with localizing mRNPs exhibiting a strong minus-end-directed bias. Single-molecule fluorescence measurements reveal that RNA localization signals increase the average number of dynein and dynactin components recruited to individual mRNPs. We find that, surprisingly, individual RNA molecules are present in motile mRNPs in vitro and present evidence that this is also the case in vivo. Thus, RNA oligomerization is not obligatory for transport. Our findings lead to a model in which RNA localization signals produce highly polarized distributions of transcript populations through modest changes in motor copy number on single mRNA molecules.

In muscle inorganic phosphate strongly decreases force generation in the presence of millimolar MgATP, whereas phosphate slows shortening velocity only at micromolar MgATP concentrations. It is still controversial whether reduction in shortening velocity by phosphate results from phosphate binding to the nucleotide-free myosin head or from binding of phosphate to an actomyosin-ADP state as postulated for the inhibition of force generation by phosphate. Because most single-molecule studies are performed at micromolar concentrations of MgATP where phosphate effects on movement are rather prominent, clarification of the mechanisms of phosphate inhibition is essential for interpretation of data in which phosphate is used in single molecule studies to probe molecular events of force generation and movement. In in vitro assays we found that inhibition of filament gliding by inorganic phosphate was associated with increased fragmentation of actin filaments. In addition, phosphate did not extend dwell times Muscle contraction involves the sliding of actin filaments relative to myosin filaments (1, 2), which is driven by cyclic interactions of the myosin head domains with the actin filaments, powered by hydrolysis of ATP. During each ATP-hydrolysis cycle chemical energy of ATP hydrolysis is transformed into mechanical work by a multistep power-stroke which drives the actin filaments several nanometers past the myosin filaments. The power-stroke is associated with the release of the ATP hydrolysis products, P i (inorganic phosphate) and ADP, from the active site (3). It is generally believed that P i release from the AM⅐ADP⅐P i 3 complex is closely related to the initiation of the power-stroke (4) and to the transition of the myosin head domain from states of weak and non-stereospecific actin binding to states of strong and stereospecific binding to actin (5-7). Because of the assumed close relation between power stroke and the release of inorganic phosphate from the myosin head domain, studying the effects of inorganic phosphate on contracting muscle fibers became a main element to elucidate the relation between P i release and force generation. Effects of P i were examined on isometric force (4, 8 -14) and unloaded shortening velocity (9, 13, 15) as well as on force transients in response to release of P i from caged P i (16,17). As a result of these studies it was proposed that inhibition of active force by inorganic phosphate results from rebinding of P i to the AM⅐ADP intermediate that is formed after P i release (cf. Scheme 1). Thus, the release of phosphate is reversed and a strong binding force generating cross-bridges in the AM⅐ADP state are reversed to a weak binding non-force generating AM⅐ADP⅐P i state (the AM⅐ADP⅐Pi I state in Scheme 1) that is in rapid equilibrium with the detached M⅐ADP⅐P i I intermediate. To fully account for the observed steady state kinetic data and the force transients recorded upon release of P i from caged P i , it was proposed that a strong binding AM⅐ADP⅐P i intermediate (the AM⅐AD...

Function of Mark4 −/− hearts after myocardial infarctionTo evaluate the effect of MARK4 in the setting of ischaemic heart disease, we used a mouse model of permanent left anterior descending

Coupling of ATP hydrolysis to structural changes in the motor domain is fundamental to the driving of motile functions by myosins. Current understanding of this chemomechanical coupling is primarily based on ensemble average measurements in solution and muscle fibers. Although important, the averaging could potentially mask essential details of the chemomechanical coupling, particularly for mixed populations of molecules. Here, we demonstrate the potential of studying individual myosin molecules, one by one, for unique insights into established systems and to dissect mixed populations of molecules where separation can be particularly challenging. We measured ATP turnover by individual myosin molecules, monitoring appearance and disappearance of fluorescent spots upon binding/dissociation of a fluorescent nucleotide to/from the active site of myosin. Surprisingly, for all myosins tested, we found two populations of fluorescence lifetimes for individual myosin molecules, suggesting that termination of fluorescence occurred by two different paths, unexpected from standard kinetic schemes of myosin ATPase. In addition, molecules of the same myosin isoform showed substantial intermolecular variability in fluorescence lifetimes. From kinetic modeling of our two fluorescence lifetime populations and earlier solution data, we propose two conformers of the active site of myosin, one that allows the complete ATPase cycle and one that dissociates ATP uncleaved. Statistical analysis and Monte Carlo simulations showed that the intermolecular variability in our studies is essentially due to the stochastic behavior of enzyme kinetics and the limited number of ATP binding events detectable from an individual myosin molecule with little room for static variation among individual molecules, previously described for other enzymes.single ATP turnover assay | ATP dwell times | dwell time distribution | TIRF microscopy F orces and movements, generated when myosins interact with actin filaments, are driven by the coupling of conformational changes in the myosin head domain to particular steps of the ATP hydrolysis cycle (1-3). Essential steps of the ATP hydrolysis cycle for skeletal muscle myosin and acto-myosin were characterized in solution studies (1,4,5). With the concept of Hill (6), it became possible to relate solution studies to mechanical, biochemical, and structural studies on muscle fibers (7, 8) to form a general concept for the coupling of ATP hydrolysis to the generation of mechanical work (6, 9).In studies on large ensembles of myosin molecules such as solution and fiber studies, however, crucial reaction steps could be masked by the ensemble averaging and thus may complicate relating solution kinetics to structural changes and the generation of forces and movements. In addition, ensemble studies can be complicated by mixed populations of myosin molecules-for example, by the presence of different myosin isoforms or different posttranslational modifications. Here we explored the feasibility to study ATPase kinetics from ...