Functional and Structural Properties of Cytoplasmic Tropomyosin Isoforms Tpm1.8 and Tpm1.9.

The actin cytoskeleton is one of the most important players in cell motility, adhesion, division, and functioning. The regulation of specific microfilament formation largely determines cellular functions. The main actin-binding protein in animal cells is tropomyosin (Tpm). The unique structural and functional diversity of microfilaments is achieved through the diversity of Tpm isoforms. In our work, we studied the properties of the cytoplasmic isoforms Tpm1.8 and Tpm1.9. The results showed that these isoforms are highly thermostable and differ in the stability of their central and C-terminal fragments. The properties of these isoforms were largely determined by the 6th exons. Thus, the strength of the end-to-end interactions, as well as the affinity of the Tpm molecule for F-actin, differed between the Tpm1.8 and Tpm1.9 isoforms. They were determined by whether an alternative internal exon, 6a or 6b, was included in the Tpm isoform structure. The strong interactions of the Tpm1.8 and Tpm1.9 isoforms with F-actin led to the formation of rigid actin filaments, the stiffness of which was measured using an optical trap. It is quite possible that the structural and functional features of the Tpm isoforms largely determine the appearance of these isoforms in the rigid actin structures of the cell cortex.

Structural and Functional Properties of Tropomyosin Isoforms Tpm4.1 and Tpm2.1.

Tropomyosin (Tpm) is one of the most important partners of actin filament that largely determines its properties. In animal organisms, there are different isoforms of Tpm, which are believed to be involved in the regulation of various cellular functions. However, molecular mechanisms by which various Tpm cytoplasmic regulate of the functioning of actin filaments are still poorly understood. Here, we investigated the properties of Tpm2.1 and Tpm4.1 isoforms and compared them to each other and to more extensively studied Tpm isoforms. Tpm2.1 and Tpm4.1 were very similar in their affinity to F-actin, thermal stability, and resistance to limited proteolysis by trypsin, but differed markedly in the viscosity of their solutions and thermal stability of their complexes with F-actin. The main difference of Tpm2.1 and Tpm4.1 from other Tpm isoforms (e.g., Tpm1.6 and Tpm1.7) was their extremely low thermal stability as measured by the CD and DSC methods. We suggested the possible causes of this instability based on comparing the amino acid sequences of Tpm4.1 and Tpm2.1 with the sequences of Tpm1.6 and Tpm1.7 isoforms, respectively, that have similar exon structure.

A census of actin-associated proteins in humans.

Actin filaments help in maintaining the cell structure and coordinating cellular movements and cargo transport within the cell. Actin participates in the interaction with several proteins and also with itself to form the helical filamentous actin (F-actin). Actin-binding proteins (ABPs) and actin-associated proteins (AAPs) coordinate the actin filament assembly and processing, regulate the flux between globular G-actin and F-actin in the cell, and help maintain the cellular structure and integrity. We have used protein-protein interaction data available through multiple sources (STRING, BioGRID, mentha, and a few others), functional annotation, and classical actin-binding domains to identify actin-binding and actin-associated proteins in the human proteome. Here, we report 2482 AAPs and present an analysis of their structural and sequential domains, functions, evolutionary conservation, cellular localization, abundance, and tissue-specific expression patterns. This analysis provides a base for the characterization of proteins involved in actin dynamics and turnover in the cell.

Effects of environmental stress factors on the actin cytoskeleton of fungi and plants: Ionizing radiation and ROS.

Actin is an abundant and multifaceted protein in eukaryotic cells that has been detected in the cytoplasm as well as in the nucleus. In cooperation with numerous interacting accessory-proteins, monomeric actin (G-actin) polymerizes into microfilaments (F-actin) which constitute ubiquitous subcellular higher order structures. Considering the extensive spatial dimensions and multifunctionality of actin superarrays, the present study analyses the issue if and to what extent environmental stress factors, specifically ionizing radiation (IR) and reactive oxygen species (ROS), affect the cellular actin-entity. In that context, this review particularly surveys IR-response of fungi and plants. It examines in detail which actin-related cellular constituents and molecular pathways are influenced by IR and related ROS. This comprehensive survey concludes that the general integrity of the total cellular actin cytoskeleton is a requirement for IR-tolerance. Actin's functions in genome organization and nuclear events like chromatin remodeling, DNA-repair, and transcription play a key role. Beyond that, it is highly significant that the macromolecular cytoplasmic and cortical actin-frameworks are affected by IR as well. In response to IR, actin-filament bundling proteins (fimbrins) are required to stabilize cables or patches. In addition, the actin-associated factors mediating cellular polarity are essential for IR-survivability. Moreover, it is concluded that a cellular homeostasis system comprising ROS, ROS-scavengers, NADPH-oxidases, and the actin cytoskeleton plays an essential role here. Consequently, besides the actin-fraction which controls crucial genome-integrity, also the portion which facilitates orderly cellular transport and polarized growth has to be maintained in order to survive IR.

Smooth Muscle Myosin Localizes at the Leading Edge and Regulates the Redistribution of Actin-regulatory Proteins during Migration.

Airway smooth muscle cell migration plays an essential role in airway development, repair, and remodeling. Smooth muscle myosin II has been traditionally thought to localize in the cytoplasm solely and regulates cell migration by affecting stress fiber formation and focal adhesion assembly. In this study, we unexpectedly found that 20-kDa myosin light chain (MLC20) and myosin-11 (MYH11), important components of smooth muscle myosin, were present at the edge of lamellipodia. The knockdown of MLC20 or MYH11 attenuated the recruitment of c-Abl, cortactinProfilin-1 (Pfn-1), and Abi1 to the cell edge. Moreover, myosin light chain kinase (MLCK) colocalized with integrin ß1 at the tip of protrusion. The inhibition of MLCK attenuated the recruitment of c-Abl, cortactin, Pfn-1, and Abi1 to the cell edge. Furthermore, MLCK localization at the leading edge was reduced by integrin ß1 knockdown. Taken together, our results demonstrate that smooth muscle myosin localizes at the leading edge and orchestrates the recruitment of actin-regulatory proteins to the tip of lamellipodia. Mechanistically, integrin ß1 recruits MLCK to the leading edge, which catalyzes MLC20 phosphorylation. Activated myosin regulates the recruitment of actin-regulatory proteins to the leading edge, and promotes lamellipodial formation and migration.

Mechanical stress effects on transcriptional regulation of genes encoding microtubule- and actin-associated proteins.

Plant cytoskeleton regulation has been studied using a new approach based on both (1) pharmacological analysis of tubulin and actin inhibitors and (2) mechanical stimulation achieved by using a slow-rotating (2 rpm) clinostat in combination with transcriptional analysis of genes encoding TUA6, ACT2, MAP65-1, CLASP, PLDδ, FH4 and FH1 proteins in Arabidopsis thaliana seedling roots. The obtained data suggest feedback between the organization of microtubule (MT) and actin filament (AF) networks and the expression of the ACT2, TUA6, MAP65-1, CLASP and FH1/FH4 genes. Different regulation of feedback between MT/AF organization and TUA6, ACT2, MAP65-1, CLASP, FH4 and FH1 gene expression was noted during slow clinorotation, possibly due to altered mechanical impact on the cortical cytoskeleton. For the first time, the expression of the tubulin-associated gene MAP65-1 was shown to be dependent upon the organization of AFs. TUA6, MAP65-1, CLASP, FH1 and FH4 likely participate in mechanical signal transduction. Our work demonstrated that slow clinorotation is able to cause mechanical stress.

Actin-Associated Proteins and Small Molecules Targeting the Actin Cytoskeleton.

Actin-associated proteins (AAPs) act on monomeric globular actin (G-actin) and polymerized filamentous actin (F-actin) to regulate their dynamics and architectures which ultimately control cell movement, shape change, division; organelle localization and trafficking. Actin-binding proteins (ABPs) are a subset of AAPs. Since actin was discovered as a myosin-activating protein (hence named actin) in 1942, the protein has also been found to be expressed in non-muscle cells, and numerous AAPs continue to be discovered. This review article lists all of the AAPs discovered so far while also allowing readers to sort the list based on the names, sizes, functions, related human diseases, and the dates of discovery. The list also contains links to the UniProt and Protein Atlas databases for accessing further, related details such as protein structures, associated proteins, subcellular localization, the expression levels in cells and tissues, mutations, and pathology. Because the actin cytoskeleton is involved in many pathological processes such as tumorigenesis, invasion, and developmental diseases, small molecules that target actin and AAPs which hold potential to treat these diseases are also listed.