IntAct: a non-disruptive internal tagging strategy to study actin isoform organization and function

Actin plays a central role in many biological processes such as cell division, motility and contractility. In birds and mammals, actin has six, highly conserved isoforms, four of which are primarily present in muscles and two that are ubiquitously expressed across tissues. While each isoform has non-redundant biological functions, we currently lack the tools to investigate the molecular basis for isoform specificity due to their high similarity and the limited possibilities to manipulate actin. To solve this technical challenge, we developed IntAct, an internally tagged actin system to study actin isoform organization in fixed and living cells. For this, we performed a microscopy-based screen for 11 internal actin positions and identified one residue pair that allows for non-disruptive epitope tag integration. Using knockin cell lines with tags into the ubiquitously expressed β-actin, we demonstrate that IntAct actins are properly expressed and that their incorporation into filaments is indistinguishable from wildtype. We further show that IntAct actins can be visualized in living cells by exploiting the nanobody-targeted ALFA tag and that they keep their ability to interact with the actin binding proteins profilin and cofilin. Lastly, we also introduced the tag in the ubiquitously expressed γ-actin and demonstrate that the differential localization observed for actin isoforms remains unaltered for the IntAct actins. Together, our data demonstrate that IntAct is a versatile tool to study actin isoform localization, dynamics and molecular interactions to finally enable the molecular characterization of actin isoforms in biological processes.

Tropomyosin isoforms segregate into distinct clusters on single actin filaments

Tropomyosins (Tpm) are rod-shaped proteins that interact head-to-tail to form a continuous polymer along both sides of most cellular actin filaments. Head-to-tail interaction between adjacent Tpm molecules and the formation of an overlap complex between them leads to the assembly of actin filaments with one type of Tpm isoform in time and space. Variations in the affinity of tropomyosin isoforms for different actin structures are proposed as a potential sorting mechanism. However, the detailed mechanisms of spatio-temporal sorting of Tpms remain elusive. In this study, we investigated the early intermediates during actin-tropomyosin filament assembly, using skeletal/cardiac Tpm isoform (Tpm1.1) and a cytoskeletal isoform (Tpm1.6) that differ only in the last 27 amino acids. We investigated how the muscle isoform Tpm1.1 and the cytoskeletal isoform Tpm1.6 nucleate domains on the actin filament and tested whether (1) recruitment is affected by the actin isoform (muscle vs cytoskeletal) and (2) whether there is specificity in recruiting the same isoform to a domain at these early stages. To address these questions, actin filaments were exposed to low concentrations of fluorescent tropomyosins in solution. The filaments were immobilized onto glass coverslips and the pattern of decoration was visualized by TIRF microscopy. We show that at the early assembly stage, tropomyosins formed multiple distinct fluorescent domains (here termed "cluster") on the actin filaments. An automated image analysis algorithm was developed and validated to identify clusters and estimate the number of tropomyosins in each cluster. The analysis showed that tropomyosin isoform sorting onto an actin filament is unlikely to be driven by a preference for nucleating on the corresponding muscle or cytoskeletal actin isoforms but rather is facilitated by a higher probability of incorporating the same tropomyosin isoforms into an early assembly intermediate. We showed that the 27 amino acids at the end of each tropomyosin seem to provide enough molecular information for attachment of the same tropomyosin isoforms adjacent to each other on an actin filament. This results in the formation of homogeneous clusters composed of the same isoform rather than clusters with mixed isoforms.

Inhibition of polar actin assembly by astral microtubules is required for cytokinesis

During cytokinesis, the actin cytoskeleton is partitioned into two spatially distinct actin isoform specific networks: a β-actin network that generates the equatorial contractile ring, and a γ-actin network that localizes to the cell cortex. Here we demonstrate that the opposing regulation of the β- and γ-actin networks is required for successful cytokinesis. While activation of the formin DIAPH3 at the cytokinetic furrow underlies β-actin filament production, we show that the γ-actin network is specifically depleted at the cell poles through the localized deactivation of the formin DIAPH1. During anaphase, CLIP170 is delivered by astral microtubules and displaces IQGAP1 from DIAPH1, leading to formin autoinhibition, a decrease in cortical stiffness and localized membrane blebbing. The contemporaneous production of a β-actin contractile ring at the cell equator and loss of γ-actin from the poles is required to generate a stable cytokinetic furrow and for the completion of cell division.

The origin of the expressed retrotransposed gene ACTBL2 and its influence on human melanoma cells’ motility and focal adhesion formation

We have recently found that β-actin-like protein 2 (actbl2) forms complexes with gelsolin in human melanoma cells and can polymerize. Phylogenetic and bioinformatic analyses showed that actbl2 has a common origin with two non-muscle actins, which share a separate history from the muscle actins. The actin groups’ divergence started at the beginning of vertebrate evolution, and actbl2 actins are characterized by the largest number of non-conserved amino acid substitutions of all actins. We also discovered that ACTBL2 is expressed at a very low level in several melanoma cell lines, but a small subset of cells exhibited a high ACTBL2 expression. We found that clones with knocked-out ACTBL2 (CR-ACTBL2) or overexpressing actbl2 (OE-ACTBL2) differ from control cells in the invasion, focal adhesion formation, and actin polymerization ratio, as well as in the formation of lamellipodia and stress fibers. Thus, we postulate that actbl2 is the seventh actin isoform and is essential for cell motility.

Knockout of ACTB and ACTG1 with CRISPR/Cas9(D10A) Technique Shows that Non-Muscle β and γ Actin Are Not Equal in Relation to Human Melanoma Cells’ Motility and Focal Adhesion Formation

Non-muscle actins have been studied for many decades; however, the reason for the existence of both isoforms is still unclear. Here we show, for the first time, a successful inactivation of the ACTB (CRISPR clones with inactivated ACTB, CR-ACTB) and ACTG1 (CRISPR clones with inactivated ACTG1, CR-ACTG1) genes in human melanoma cells (A375) via the RNA-guided D10A mutated Cas9 nuclease gene editing [CRISPR/Cas9(D10A)] technique. This approach allowed us to evaluate how melanoma cell motility was impacted by the lack of either β actin coded by ACTB or γ actin coded by ACTG1. First, we observed different distributions of β and γ actin in the cells, and the absence of one actin isoform was compensated for via increased expression of the other isoform. Moreover, we noted that γ actin knockout had more severe consequences on cell migration and invasion than β actin knockout. Next, we observed that the formation rate of bundled stress fibers in CR-ACTG1 cells was increased, but lamellipodial activity in these cells was impaired, compared to controls. Finally, we discovered that the formation rate of focal adhesions (FAs) and, subsequently, FA-dependent signaling were altered in both the CR-ACTB and CR-ACTG1 clones; however, a more detrimental effect was observed for γ actin-deficient cells. Our research shows that both non-muscle actins play distinctive roles in melanoma cells’ FA formation and motility.

The elusive actin cytoskeleton of a green alga expressing both conventional and divergent actins

The green alga Chlamydomonas reinhardtii is a leading model system to study photosynthesis, cilia, and the generation of biological products. The cytoskeleton plays important roles in all of these cellular processes, but to date, the filamentous actin network within Chlamydomonas has remained elusive. By optimizing labeling conditions, we can now visualize distinct linear actin filaments at the posterior of the nucleus in both live and fixed vegetative cells. Using in situ cryo-electron tomography, we confirmed this localization by directly imaging actin filaments within the native cellular environment. The fluorescently labeled structures are sensitive to the depolymerizing agent latrunculin B (Lat B), demonstrating the specificity of our optimized labeling method. Interestingly, Lat B treatment resulted in the formation of a transient ring-like filamentous actin structure around the nucleus. The assembly of this perinuclear ring is dependent upon a second actin isoform, NAP1, which is strongly up-regulated upon Lat B treatment and is insensitive to Lat B–induced depolymerization. Our study combines orthogonal strategies to provide the first detailed visual characterization of filamentous actins in Chlamydomonas, allowing insights into the coordinated functions of two actin isoforms expressed within the same cell.

The elusive actin cytoskeleton of a green alga expressing both conventional and divergent actins

The green alga Chlamydomonas reinhardtii is a leading model system to study photosynthesis, cilia, and the generation of biological products. The cytoskeleton plays important roles in all of these cellular processes, but to date, the filamentous actin network within Chlamydomonas has remained elusive. By optimizing labeling conditions, we can now visualize distinct linear actin filaments at the posterior of the nucleus in both live and fixed vegetative cells. Using in situ cryo-electron tomography, we confirmed this localization by directly imaging actin filaments within the native cellular environment. The fluorescently-labeled structures are sensitive to the depolymerizing agent Latrunculin B (Lat B), demonstrating the specificity of our optimized labeling method. Interestingly, Lat B treatment resulted in the formation of a transient ring-like filamentous actin structure around the nucleus. The assembly of this perinuclear ring is dependent upon a second actin isoform, NAP1, which is strongly upregulated upon Lat B treatment and is insensitive to Lat B-induced depolymerization. Our study combines orthogonal strategies to provide the first detailed visual characterization of filamentous actins in Chlamydomonas, allowing insights into the coordinated functions of two actin isoforms expressed within the same cell.

β-Actin shows limited mobility and is required only for supraphysiological insulin-stimulated glucose transport in young adult soleus muscle

Studies in skeletal muscle cell cultures suggest that the cortical actin cytoskeleton is a major requirement for insulin-stimulated glucose transport, implicating the β-actin isoform, which in many cell types is the main actin isoform. However, it is not clear that β-actin plays such a role in mature skeletal muscle. Neither dependency of glucose transport on β-actin nor actin reorganization upon glucose transport have been tested in mature muscle. To investigate the role of β-actin in fully differentiated muscle, we performed a detailed characterization of wild type and muscle-specific β-actin knockout (KO) mice. The effects of the β-actin KO were subtle; however, we confirmed the previously reported decline in running performance of β-actin KO mice compared with wild type during repeated maximal running tests. We also found insulin-stimulated glucose transport into incubated muscles reduced in soleus but not in extensor digitorum longus muscle of young adult mice. Contraction-stimulated glucose transport trended toward the same pattern, but the glucose transport phenotype disappeared in soleus muscles from mature adult mice. No genotype-related differences were found in body composition or glucose tolerance or by indirect calorimetry measurements. To evaluate β-actin mobility in mature muscle, we electroporated green fluorescent protein (GFP)-β-actin into flexor digitorum brevis muscle fibers and measured fluorescence recovery after photobleaching. GFP-β-actin showed limited unstimulated mobility and no changes after insulin stimulation. In conclusion, β-actin is not required for glucose transport regulation in mature mouse muscle under the majority of the tested conditions. Thus, our work reveals fundamental differences in the role of the cortical β-actin cytoskeleton in mature muscle compared with cell culture.