Bacterial actin-like proteins such as MreB and FtsA have been identified through structural homology to monomeric actin and their ability to form actin-like filaments. Furthermore, recent advances in structural biology have unearthed proteins that regulate the dynamics of these bacterial actin homologs much like their eukaryotic counterparts. However, questions remain as to how the actin and actin-like proteins evolved to participate in similar functions of cell division and maintenance of cell shape. To this effect, we asked whether the bacterial and eukaryotic actin homologs share a common interactome. Using previously published literature on functional homology followed by structural alignment of functional homologs, we identified 64 unique genetic and physical interactors of actin and their corresponding 38 homologs in bacteria that also interact with MreB/FtsA. We also report for the first time the identification of 10 bacterial homologs of 14 known actin interactors. Additionally, we reasoned that the homologous interactors would be essential to the function of actin. Therefore, the homologous interactors may show a high degree of co-expression with actin during cell division and differentiation, processes essential to embryogenesis. Therefore, we analysed the single-cell RNA expression data of early stages of human embryogenesis. We observed that ~66% of the homologous actin interactors showed positive correlation with the expression of beta-actin during early human embryogenesis. Taken together, our study not just enlists a common actin/MreB interactome, but also highlights the physiological significance of these interactions.
Intracellular transport is essential for cellular organization and function. Kinesin-1, the founding member of the kinesin superfamily, delivers a wide range of cargoes along microtubules. Since its discovery, extensive work has identified cargo-kinesin interactions and regulatory mechanisms. Yet a basic question remains: why does kinesin-driven transport in cells often appear faster than the single-molecule velocity of kinesin-1 measured in vitro? Cargoes moved by kinesin-1 can exhibit markedly higher velocities in cells than isolated kinesin-1 molecules under commonly used assay conditions. How can kinesin-1-driven transport be accelerated in the cytoplasm crowded with macromolecules and other structures? In this Perspective, we discuss potential mechanisms underlying this "kinesin speed paradox" and consider what they reveal about motor behavior in complex intracellular milieus.
The simple nematode Caenorhabditis elegans has long served as a powerful genetic model system for studying muscle structure and function, with sarcomeres of the striated body wall muscle (BWM) that are considered largely homologous to those in vertebrates. However, we have observed a population of F-actin in BWM separate from I-bands and centered at the sarcomere M-line, something not seen in vertebrate sarcomeres. Here, we investigate whether M-line-associated F-actin is a true feature of worm muscle, or an artifact of sample preparation. Using thin filament components tagged with fluorescent proteins, we confirm that living C. elegans BWM has a traditional sarcomere organization with actin restricted to I-bands, whereas two methods of fixation resulted in varying degrees of mislocalization of actin as well as barbed end capping protein/CapZ and tropomodulin. Additionally, we observe in live animals that α-actinin-rich dense bodies are flanked by discrete patches of CapZ, something we cannot observe in fixed animals, but which provides some support for a model in which dense bodies anchor thin filaments in a manner similar to vertebrate Z-discs. Overall, our work highlights the susceptibility of thin filaments in worm BWM to fixation artifacts and the potential utility in studying sarcomere structure in live animals.
Mammals have four tropomyosin (TPM) genes (TPM1, TPM2, TPM3, and TPM4) that produce various isoforms through alternative splicing. TPM1 generates both myofibrillar (Tpm1.1, Tpm1.2) and non-myofibrillar isoforms, including those with an exon 9d peptide or with an exon 9a peptide. These non-myofibrillar isoforms are involved in regulating actin-based structures and processes in non-muscle cells. This study focused on the expression of the non-myofibrillar HMW Tpm1 isoforms (Tpm1.3, Tpm1.4, Tpm1.5, Tpm1.6, Tpm1.7, and Tpm1.14) during human inducible pluripotent stem cells (hiPSC) differentiation into cardiomyocytes (CMs) at different time points (Days 0, 5, 10, 15, 20). We have determined the expression of various Tpm1 transcripts by qRT-PCR using isoform-specific primer-pairs. Western blotting with Tpm1-exon 6a and Tpm1-exon 9d antibodies and 2D Western blotting with Tpm1-exon 6a antibody followed by mass spectra analyses were used to evaluate protein expression. Transcripts of non-myofibrillar Tpm1 isoforms peaked at Day 15 and continued at a slightly lower level in mature hiPSC-CM until Day 20. However, no expression of Tpm1.3 or Tpm1.14 has been observed. Protein expression of Tpm1.4, Tpm1.5, Tpm1.6, and Tpm1.7 increases up to Day 15 but practically disappears by Day 20 hiPSC-CM, suggesting their production is decreased, or more likely they are degraded intracellularly. The results suggest that the proteins have the potential to be transiently involved in the early stages of CM differentiation. This study also demonstrates that Tpm1.5, when fused with YFP in an expression construct and transfected into embryonic chicken CM and Day 20 mature hiPSC-CMs, can be organized into cardiac myofibrils despite being previously characterized as a non-muscle isoform. Our observation is further substantiated by the fact that YFP-Tpm1.5 fusion protein can be ectopically expressed and incorporated into the myofibrils of chicken myotube skeletal muscle, which is known to be more stringent than cardiac muscle with regards to myofibril remodeling. Paradoxically, anti-6a antibody fails to recognize the organized YFP-Tpm1.5 fusion protein in embryonic chicken CMs, embryonic chicken skeletal muscle myotubes, or in Day 20 mature hiPSC-CMs. Interestingly, the antibody recognizes the denatured YFP-Tpm1 fusion protein in Western blot analyses. It is well-documented in the literature that an antibody epitope may fail to recognize its antigen when the antigen is in its native state in living cells, often because the epitope is buried, altered conformationally, or inaccessible. Conversely, while the Tpm1.Ex6a antibody stains Tpm1.5 in cell nuclei, the YFP-Tpm1.5 fusion protein does not localize to the cell nuclei. This suggests conformational differences between nuclear and cytoplasmic Tpm1 protein(s) and differential access of the fusion protein to different cellular locations. This also suggests that it is endogenous Tpm1 6a-containing protein identified in the nuclei.
