Actin Cytoskeleton

Bleb formation has been correlated with nonmuscle myosin II (NM-II) activity. Whether three isoforms of NM-II (NM-IIA, -IIB, and -IIC) have the same or differential roles in bleb formation is not well understood. Here, we report that ectopically expressed GFP-tagged NM-II isoforms exhibit different types of membrane protrusions such as multiple blebs, lamellipodia, combination of both or absence of any such protrusions in MCF-7 cells. Quantification suggests that 50% of NM-IIA-GFP, 29% of NM-IIB-GFP and 19% of NM-IIC1-GFP expressing MCF-7 cells show multiple blebs formation compared with 36% of GFP alone expressing cells. Interestingly, NM-IIB has almost 50% lesser rate of dissociation from actin filament compared with NM-IIA and -IIC1 as determined by FRET analysis both at cell and bleb cortices. When we induce bleb formation by disruption of the cortex, we find that all three NM-II-GFP isoforms can re-appear and form filaments to a different degree in the growing bleb. NM-IIB-GFP ...

Linearolactone (1) and kaempferol (2) have amebicidal activity in in vitro studies. The type of cell death induced by 1 and 2 and their effects on the virulence of E. histolytica were analyzed by transmission and confocal electron microscopy, reactive oxygen species (ROS) production, and apoptosis, detected by flow cytometry with dichlorofluorescein 2′,7′-diacetate and annexin-V binding, respectively, and confirmed by TUNEL. The interaction of 1 and 2 with actin was analyzed by docking, and the in vivo amoebicidal activity was established with the Mesocricetus auratus model; amebic liver abscess (ALA) development was evaluated by magnetic resonance (MR) and validated post mortem. In vitro, compounds 1 and 2 caused chromatin condensation, intracellular ROS, and loss of actin structures. Coupling analysis showed that they bind to the allosteric and catalytic sites of actin with binding energies of −11.30 and −8.45 kcal/mol, respectively. Treatments with 1 and 2 induced a decrease in ALA formation without toxic effects on the liver and kidney. Thus, compound 1, but not 2, was able to induce apoptosis-like effects in E. histolytica trophozoites by intracellular production of ROS that affected the actin cytoskeleton structuration. In vivo, compound 1 was more active than compound 2 to reduce the development of ALA. A mebiasis, caused by Entamoeba histolytica, is acquired after ingesting food or water contaminated with cysts, which excyst and give rise to trophozoites, which grow in the intestine. Once it is colonized, trophozoites invade the tissue and migrate to different organs, such as the liver. To date, the most commonly used treatment is metronidazole. However, studies have demonstrated the development of resistance to this drug, both in vitro and in vivo. 1 Therefore, there is a need to search for new, safer, and more effective antiamoebic drugs. Linearolactone (1) is a neo-clerodane diterpenoid isolated from Salvia polystachya, 2 and kaempferol (2) 3 is a flavonol isolated from Cuphea pinetorum, which have been characterized previously. Their antiprotozoal activities has been demonstrated in vitro, 2 where the half-maximal inhibitory concentrations (IC 50) of these compounds were established against Giardia lamblia and E. histolytica. Recently, it was reported that 2 inhibits the growth of E. histolytica by altering the functions of the cytoskeleton. 4 Based on these reports, 1 and 2 are considered good candidates to develop antiamoebic treatments. 3 However, the action mechanism of these specialized metabolites in animal models has not been described. In this work, we determined the type of death induced by 1 and 2 on E. histolytica trophozoites. The antiamoebic effect of both compounds on the development of amoebic liver abscess in a Mesocricetus auratus model was also analyzed, 5 and the potential toxic effects during the administration of the treatments were also examined.

The inherited muscle diseases, skeletal muscle nemaline myopathy and cardiac muscle hypertrophic myopathy (HCM) have been recognised for decades. Recently it has become apparent that mutations in almost any protein component of the sarcomere could cause myopathy. Thus changes in many sarcomeric protein genes can produce a common phenotype. Several recent publications indicate the opposite property: mutations in one sarcomeric protein can produce different muscle disease phenotypes. The most dramatic example of this property is actin, mutations in which are associated with hypertrophic cardiomyopathy, dilated cardiomyopathy, nemaline myopathy and actin myopathy.

▪   Many pathogens actively exploit the actin cytoskeleton during infection. This exploitation may take place during entry into mammalian cells after engagement of a receptor and/or as series of signaling events culminating in the engulfment of the microorganism. Although actin rearrangements are a common feature of most internalization events (e.g. entry of Listeria, Salmonella, Shigella, Yersinia, Neisseria, and Bartonella), bacterial and other cellular factors involved in entry are specific to each bacterium. Another step during which pathogens harness the actin cytoskeleton takes place in the cytosol, within which some bacteria (Listeria, Shigella, Rickettsia) or viruses (vaccinia virus) are able to move. Movement is coupled to a polarized actin polymerization process, with the formation of characteristic actin tails. Increasing attention has focused on this phenomenon due to its striking similarity to cellular events occurring at the leading edge of locomoting cells. Thus patho...

Motile growth cones lead growing axons through developing tissues to synaptic targets. These behaviors depend on the organization and dynamics of actin filaments that fill the growth cone leading margin (peripheral (P-) domain). Actin filament organization in growth cones is regulated by actin-binding proteins that control all aspects of filament assembly, turnover, interactions with other filaments and cytoplasmic components, and participation in producing mechanical forces. Actin filament polymerization drives protrusion of sensory filopodia and lamellipodia, and actin filament connections to the plasma membrane link the filament network to adhesive contacts of filopodia and lamellipodia with other surfaces. These contacts stabilize protrusions and transduce mechanical forces generated by actomyosin activity into traction that pulls an elongating axon along the path towards its target. Adhesive ligands and extrinsic guidance cues bind growth cone receptors and trigger signaling ac...

ObjectiveMutations in the α-skeletal actin gene (ACTA1) result in a variety of inherited muscle disorders characterized by different pathologies and variable clinical phenotypes. Mutations at Val163 in ACTA1 result in pure intranuclear rod myopathy; however, the molecular mechanisms by which mutations at Val163 lead to intranuclear rod formation and muscle weakness are unknown.Mutations in the α-skeletal actin gene (ACTA1) result in a variety of inherited muscle disorders characterized by different pathologies and variable clinical phenotypes. Mutations at Val163 in ACTA1 result in pure intranuclear rod myopathy; however, the molecular mechanisms by which mutations at Val163 lead to intranuclear rod formation and muscle weakness are unknown.Methods and ResultsWe investigated the effects of the Val163Met mutation in ACTA1 in tissue culture and Drosophila models, and in patient muscle. In cultured cells, the mutant actin tends to aggregate rather than incorporate into cytoplasmic microfilaments, and it affects the dynamics of wild-type actin, causing it to accumulate with the mutant actin in the nucleus. In Drosophila, the Val163Met mutation severely disrupts the structure of the muscle sarcomere. The intranuclear aggregates in patient muscle biopsies impact on nuclear structure and sequester normal Z-disc–associated proteins within the nucleus; however, the sarcomeric structure is relatively well preserved, with evidence of active regeneration. By mass spectrometry, the levels of mutant protein are markedly reduced in patient muscle compared with control.We investigated the effects of the Val163Met mutation in ACTA1 in tissue culture and Drosophila models, and in patient muscle. In cultured cells, the mutant actin tends to aggregate rather than incorporate into cytoplasmic microfilaments, and it affects the dynamics of wild-type actin, causing it to accumulate with the mutant actin in the nucleus. In Drosophila, the Val163Met mutation severely disrupts the structure of the muscle sarcomere. The intranuclear aggregates in patient muscle biopsies impact on nuclear structure and sequester normal Z-disc–associated proteins within the nucleus; however, the sarcomeric structure is relatively well preserved, with evidence of active regeneration. By mass spectrometry, the levels of mutant protein are markedly reduced in patient muscle compared with control.InterpretationData from our tissue culture and Drosophila models show that the Val163Met mutation in α-skeletal actin can affect the dynamics of other actin isoforms and severely disrupt sarcomeric structure, processes that can contribute to muscle weakness. However, in human muscle, there is evidence of regeneration, and the mutant protein tends to aggregate rather than incorporate into cytoplasmic microfilaments in cells. These are likely compensatory processes that ameliorate the effects of the mutant actin and contribute to the milder clinical and pathological disease phenotype. Ann Neurol 2007Data from our tissue culture and Drosophila models show that the Val163Met mutation in α-skeletal actin can affect the dynamics of other actin isoforms and severely disrupt sarcomeric structure, processes that can contribute to muscle weakness. However, in human muscle, there is evidence of regeneration, and the mutant protein tends to aggregate rather than incorporate into cytoplasmic microfilaments in cells. These are likely compensatory processes that ameliorate the effects of the mutant actin and contribute to the milder clinical and pathological disease phenotype. Ann Neurol 2007

A 38-kd protein that associates with F-actin structures in activated platelets and endothelial cells was purified, cloned, and characterized. The protein contains an N-terminal PDZ motif, a large intervening sequence, and a C-terminal LIM domain and was identified as the human homolog of rat CLP36. The study showed that CLP36 associates with actin filaments and stress fibers that are formed during shape change and spreading of platelets and during migration and contraction of endothelial cells. CLP36 binds to alpha-actinin-1 as shown by coimmunoprecipitation, pull-down experiments, yeast 2-hybrid analysis, and blot overlay assays and colocalizes with alpha-actinin-1 along endothelial actin stress fibers. In contrast to alpha-actinin-1, CLP36 was absent from focal adhesions in both activated platelets and endothelial cells. The N-terminal part of CLP36 containing the PDZ domain and the intervening region, but not the LIM domain, targeted enhanced green fluorescent protein fusion prot...

The actin cytoskeleton is a primary determinant of cell shape and motility. Studies on actin regulatory proteins are now coupled with studies of the signal transduction that directs actin cytoskeleton reorganization, and we have gained insights into how external stimuli such as chemoattractants drive changes in actin cytoskeleton. Chemoattractants regulate actin regulatory proteins such as the Arp2⧸3 complex through WASP family proteins, ADF⧸cofilin downstream of LIM-kinase, and various other phosphoinositide-dependent or -independent pathways. Through branching of actin filaments, Arp2⧸3 complex-dependent actin polymerization is suffcient to generate the force necessary for protrusion.

Viral infection converts the normal functions of a cell to optimize viral replication and virion production. One striking observation of this conversion is the reconfiguration and reorganization of cellular actin, affecting every stage of the viral life cycle, from entry through assembly to egress. The extent and degree of cytoskeletal reorganization varies among different viral infections, suggesting the evolution of myriad viral strategies. In this Review, we describe how the interaction of viral proteins with the cell modulates the structure and function of the actin cytoskeleton to initiate, sustain and spread infections. The molecular biology of such interactions continues to engage virologists in their quest to understand viral replication and informs cell biologists about the role of the cytoskeleton in the uninfected cell.

We have developed a model of cardiac thin filament activation using an Ising model approach from equilibrium statistical physics. This model explicitly represents nearest-neighbor interactions between 26 troponin/tropomyosin units along a one-dimensional array that represents the cardiac thin filament. With transition rates chosen to match experimental data, the results show that the resulting force-pCa (F-pCa) relations are similar to Hill functions with asymmetries, as seen in experimental data. Specifically, Hill plots showing (log(F/(1-F)) vs. log [Ca]) reveal a steeper slope below the half activation point (Ca50) compared with above. Parameter variation studies show interplay of parameters that affect the apparent cooperativity and asymmetry in the F-pCa relations. The model also predicts that Ca binding is uncooperative for low [Ca], becomes steeper near Ca50, and becomes uncooperative again at higher [Ca]. The steepness near Ca50 mirrors the steep F-pCa as a result of thermodynamic considerations. The model also predicts that the correlation between troponin/tropomyosin units along the one-dimensional array quickly decays at high and low [Ca], but near Ca50, high correlation occurs across the whole array. This work provides a simple model that can account for the steepness and shape of F-pCa relations that other models fail to reproduce.