The ability of cells to generate forces through actin filament turnover was an early adaptation in evolution. While much is known about how filaments grow, mechanisms their disassembly are incompletely understood. best-characterized factors the cofilin family proteins, which increase cytoskeletal dynamics by severing filaments. However, mechanism severed recycled back monomeric form has remained enigmatic. We report that cyclase-associated-protein (CAP) works synergy with accelerate...

Abstract Actin polymerization powers key cellular processes, including motility, morphogenesis, and endocytosis. The actin turnover cycle depends critically on “re-charging” of ADP-actin monomers with ATP, but whether this reaction requires dedicated proteins in cells, the underlying mechanism, have remained elusive. Here we report that nucleotide exchange catalyzed by ubiquitous cytoskeletal regulator cyclase-associated protein (CAP) is critical for actin-based processes vivo. We determine...

Abstract Intracellular organization is largely mediated by actin turnover. Cellular networks continuously assemble and disassemble, while maintaining their overall appearance. This behavior, called “dynamic steady state,” allows cells to sense adapt environment. However, how structural stability can be maintained during the constant turnover of a limited monomer pool poorly understood. To answer this question, we developed an experimental system where polystyrene beads are propelled comet in...

Significance Tight regulation is required to control biochemical reactions in cells so that these can be activated precisely when and where needed. Our work focuses on actin, a protein polymerizes into filaments at least 20 distinct processes mammalian cells. We have previously shown lysine acetylation, relatively minor modification crucial for regulating specific population of actin through called INF2. Here we identify key acetylated lysines INF2 activity. mutations link 2 human diseases,...

Contractile actomyosin bundles, stress fibers, are crucial for adhesion, morphogenesis, and mechanosensing in nonmuscle cells. However, the mechanisms by which myosin II (NM-II) is recruited to those structures assembled into functional bipolar filaments have remained elusive. We report that UNC-45a a dynamic component of actin fibers functions as chaperone vivo. knockout cells display severe defects fiber assembly consequent abnormalities cell polarity, migration. Experiments combining...

Abstract Actin polymerization generates forces for cellular processes throughout the eukaryotic kingdom, but our understanding of ‘ancient’ actin turnover machineries is limited. We show that, despite > 1 billion years evolution, pathogenic Leishmania major parasite and mammalian actins share same overall fold co-polymerize with each other. Interestingly, harbors a simple actin-regulatory machinery that lacks cofilin ‘cofactors’, which accelerate filament disassembly in higher eukaryotes....

Complement is an important part of innate immunity. The alternative pathway complement activated when the main opsonin, C3b coats non-protected surfaces leading to opsonisation, phagocytosis and cell lysis. tightly controlled prevent autoactivation towards host cells. regulator factor H (FH), a soluble glycoprotein that terminates activation in multiple ways. FH recognizes via domains 19–20 (FH19-20). All microbes including Borrelia burgdorferi, causative agent Lyme borreliosis, must evade...

The actin cytoskeleton is critical for cell migration, morphogenesis, endocytosis, organelle dynamics, and cytokinesis. To support diverse cellular processes, filaments form a variety of structures with specific architectures dynamic properties. Key proteins specifying are tropomyosins. Non-muscle cells express several functionally non-redundant tropomyosin isoforms, which differentially control the interactions other proteins, including myosins ADF/cofilin, filaments. However, underlying...

Abstract Actin polymerization provides force for vital processes of the eukaryotic cell, but our understanding actin dynamics and energetics remains limited due to lack high-quality probes. Most current probes affect or its interactions with actin-binding proteins (ABPs), cannot track bound nucleotide. Here, we identify a family highly sensitive fluorescent nucleotide analogues structurally compatible actin. We demonstrate that these nucleotides bind actin, maintain functional number...

Abstract Protein engineering is one of the foundations biotechnology, used to increase protein stability, re-assign catalytic properties enzymes or interaction affinity between antibody and target. To date, strategies for have focussed on systematic, random computational methods introducing new mutations. Here, we introduce statistical approach fractional factorial design as a convenient powerful tool analysis mutations, allowing sampling large mutational space whilst minimising tests be...

Diseases caused by Leishmania and Trypanosoma parasites are a major health problem in tropical countries. Due to their complex life cycle involving both vertebrate insect hosts, >1 billion years of evolutionarily distance, the cell biology trypanosomatid exhibits pronounced differences animal cells. For example, actin cytoskeleton trypanosomatids is divergent when compared other eukaryotes. To understand how dynamics regulated parasites, we focused on central actin-binding protein profilin....

Immune evasion facilitates survival of Borrelia , leading to infections like relapsing fever and Lyme disease. Important mechanism for complement is acquisition the main host inhibitor, factor H (FH). By determining 2.2 Å crystal structure Factor binding protein A (FhbA) from hermsii in complex with FH domains 19–20, combined extensive mutagenesis, we identified structural by which B . utilizes FhbA immune evasion. Moreover, structure-guided sequence database analysis a new family...

Abstract Intracellular organization is largely mediated by the actin turnover. Cellular networks consume matter and energy to sustain their dynamics, while maintaining appearance. This behavior, called ‘dynamic steady state’, enables cells sense adapt environment. However, how structural stability can be maintained during constant turnover of a limited monomer pool poorly understood. To answer this question, we developed an experimental system using bead motility in compartment with amount...

ABSTRACT Diseases caused by Leishmania , and Trypanosoma parasites, such as leishmaniasis African sleeping sickness, are a major health problem in tropical countries. Due to their complex life cycle involving both vertebrate insect hosts, > 1 billion years of evolutionarily distance, the cell biology these trypanosomatid parasites exhibits pronounced differences animal cells. For example, actin cytoskeleton trypanosomatids is highly divergent when compared other eukaryotes. To understand...