Plectin.

Prechova et al. introduce the giant cytoskeletal crosslinker protein plectin.

Plectin-mediated cytoskeletal crosstalk controls cell tension and cohesion in epithelial sheets.

The coordinated interplay of cytoskeletal networks critically determines tissue biomechanics and structural integrity. Here, we show that plectin, a major intermediate filament-based cytolinker protein, orchestrates cortical cytoskeletal networks in epithelial sheets to support intercellular junctions. By combining CRISPR/Cas9-based gene editing and pharmacological inhibition, we demonstrate that in an F-actin-dependent context, plectin is essential for the formation of the circumferential keratin rim, organization of radial keratin spokes, and desmosomal patterning. In the absence of plectin-mediated cytoskeletal cross-linking, the aberrant keratin-desmosome (DSM)-network feeds back to the actin cytoskeleton, which results in elevated actomyosin contractility. Also, by complementing a predictive mechanical model with Förster resonance energy transfer-based tension sensors, we provide evidence that in the absence of cytoskeletal cross-linking, major intercellular junctions (adherens junctions and DSMs) are under intrinsically generated tensile stress. Defective cytoarchitecture and tensional disequilibrium result in reduced intercellular cohesion, associated with general destabilization of plectin-deficient sheets upon mechanical stress.

pyTFM: A tool for traction force and monolayer stress microscopy.

Cellular force generation and force transmission are of fundamental importance for numerous biological processes and can be studied with the methods of Traction Force Microscopy (TFM) and Monolayer Stress Microscopy. Traction Force Microscopy and Monolayer Stress Microscopy solve the inverse problem of reconstructing cell-matrix tractions and inter- and intra-cellular stresses from the measured cell force-induced deformations of an adhesive substrate with known elasticity. Although several laboratories have developed software for Traction Force Microscopy and Monolayer Stress Microscopy computations, there is currently no software package available that allows non-expert users to perform a full evaluation of such experiments. Here we present pyTFM, a tool to perform Traction Force Microscopy and Monolayer Stress Microscopy on cell patches and cell layers grown in a 2-dimensional environment. pyTFM was optimized for ease-of-use; it is open-source and well documented (hosted at https://pytfm.readthedocs.io/) including usage examples and explanations of the theoretical background. pyTFM can be used as a standalone Python package or as an add-on to the image annotation tool ClickPoints. In combination with the ClickPoints environment, pyTFM allows the user to set all necessary analysis parameters, select regions of interest, examine the input data and intermediary results, and calculate a wide range of parameters describing forces, stresses, and their distribution. In this work, we also thoroughly analyze the accuracy and performance of the Traction Force Microscopy and Monolayer Stress Microscopy algorithms of pyTFM using synthetic and experimental data from epithelial cell patches.

Mutation in PHACTR1 associated with multifocal epilepsy with infantile spasms and hypsarrhythmia.

A young boy with multifocal epilepsy with infantile spasms and hypsarrhythmia with minimal organic lesions of brain structures underwent DNA diagnosis using whole-exome sequencing. A heterozygous amino-acid substitution p.L519R in a PHACTR1 gene was identified. PHACTR1 belongs to a protein family of G-actin binding protein phosphatase 1 (PP1) cofactors and was not previously associated with a human disease. The missense single nucleotide variant in the proband was shown to occur de novo in the paternal allele. The mutation was shown in vitro to reduce the affinity of PHACTR1 for G-actin, and to increase its propensity to form complexes with the catalytic subunit of PP1. These properties are associated with altered subcellular localization of PHACTR1 and increased ability to induce cytoskeletal rearrangements. Although the molecular role of the PHACTR1 in neuronal excitability and differentiation remains to be defined, PHACTR1 has been previously shown to be involved in Slack channelopathy pathogenesis, consistent with our findings. We conclude that this activating mutation in PHACTR1 causes a severe type of sporadic multifocal epilepsy in the patient.

A low-cost uniaxial cell stretcher for six parallel wells.

Cells in the lungs, the heart, and numerous other organs, are constantly exposed to dynamic forces and deformations. To mimic these dynamic mechanical loading conditions and to study the resulting cellular responses such as morphological changes or the activation of biochemical signaling pathways, cells are typically seeded on flexible 2D substrates that are uniaxially or biaxially stretched. Here, we present an open-source cell stretcher built from parts of an Anet A8 3D printer. The cell stretcher is controlled by a fully programmable open-source software using GCode and Python. Up to six flexible optically clear substrates can be stretched simultaneously, allowing for comparative multi-batch biological studies including microscopic image analysis. The cell yield from the cell culture area of 4 cm2 per substrate is sufficient for Western-blot protein analysis. As a proof-of-concept, we study the activation of the Yes-associated protein (YAP) mechanotransduction pathway in response to increased cytoskeletal tension induced by uniaxial stretching of epithelial cells. Our data support the previously observed activation of the YAP transcription pathway by stretch-induced increase in cytoskeletal tension and demonstrate the suitability of the cell stretcher to study complex mechano-biological processes.

Molecular basis for substrate specificity of the Phactr1/PP1 phosphatase holoenzyme.

PPP-family phosphatases such as PP1 have little intrinsic specificity. Cofactors can target PP1 to substrates or subcellular locations, but it remains unclear how they might confer sequence-specificity on PP1. The cytoskeletal regulator Phactr1 is a neuronally enriched PP1 cofactor that is controlled by G-actin. Structural analysis showed that Phactr1 binding remodels PP1's hydrophobic groove, creating a new composite surface adjacent to the catalytic site. Using phosphoproteomics, we identified mouse fibroblast and neuronal Phactr1/PP1 substrates, which include cytoskeletal components and regulators. We determined high-resolution structures of Phactr1/PP1 bound to the dephosphorylated forms of its substrates IRSp53 and spectrin αII. Inversion of the phosphate in these holoenzyme-product complexes supports the proposed PPP-family catalytic mechanism. Substrate sequences C-terminal to the dephosphorylation site make intimate contacts with the composite Phactr1/PP1 surface, which are required for efficient dephosphorylation. Sequence specificity explains why Phactr1/PP1 exhibits orders-of-magnitude enhanced reactivity towards its substrates, compared to apo-PP1 or other PP1 holoenzymes.

Specific arrangements of atoms such as bulky phosphate groups can change the activity of a protein and how it interacts with other molecules. Enzymes called kinases are responsible for adding these groups onto a protein, while phosphatases remove them. Kinases are generally specific for a small number of proteins, adding phosphate groups only at sites embedded in a particular sequence in the target protein. Phosphatases, however, are generalists: only a few different types exist, which exhibit little target sequence specificity. Partner proteins can attach to phosphatases to bring the enzymes to specific locations in the cell, or to deliver target proteins to them; yet, it is unclear whether partner binding could also change the structure of the enzyme so the phosphatase can recognise only a restricted set of targets. To investigate this, Fedoryshchak, Prechová et al. studied a phosphatase called PP1 and its partner, Phactr1. First, the structure of the Phactr1/PP1 complex was examined using biochemistry approaches and X-ray crystallography. This showed that binding of Phactr1 to PP1 creates a new surface pocket, which comprised elements of both proteins. In particular, this composite pocket is located next to the part of the PP1 enzyme responsible for phosphate removal. Next, mass spectrometry and genetics methods were harnessed to identify and characterise the targets of the Phactr1/PP1 complex. Structural analysis of the proteins most susceptible to Phactr1/PP1 activity showed that they had particular sequences that could interact with Phactr1/PP1's composite pocket. Further experiments revealed that, compared to PP1 acting alone, the pocket increased the binding efficiency and reactivity of the complex 100-fold. This work demonstrates that a partner protein can make phosphatases more sequence-specific, suggesting that future studies could adopt a similar approach to examine how other enzymes in this family perform their role. In addition, the results suggest that it will be possible to design Phactr1/PP1-specific drugs that act on the composite pocket. This would represent an important proof of principle, since current phosphatase-specific drugs do not target particular phosphatase complexes.

Vimentin Intermediate Filaments as Potential Target for Cancer Treatment.

Intermediate filaments constitute the third component of the cellular skeleton. Unlike actin and microtubule cytoskeletons, the intermediate filaments are composed of a wide variety of structurally related proteins showing distinct expression patterns in tissues and cell types. Changes in the expression patterns of intermediate filaments are often associated with cancer progression; in particular with phenotypes leading to increased cellular migration and invasion. In this review we will describe the role of vimentin intermediate filaments in cancer cell migration, cell adhesion structures, and metastasis formation. The potential for targeting vimentin in cancer treatment and the development of drugs targeting vimentin will be reviewed.

Phactr1 regulates Slack (KCNT1) channels via protein phosphatase 1 (PP1).

The Slack (KCNT1) gene encodes sodium-activated potassium channels that are abundantly expressed in the central nervous system. Human mutations alter the function of Slack channels, resulting in epilepsy and intellectual disability. Most of the disease-causing mutations are located in the extended cytoplasmic C-terminus of Slack channels and result in increased Slack current. Previous experiments have shown that the C-terminus of Slack channels binds a number of cytoplasmic signaling proteins. One of these is Phactr1, an actin-binding protein that recruits protein phosphatase 1 (PP1) to certain phosphoprotein substrates. Using co-immunoprecipitation, we found that Phactr1 is required to link the channels to actin. Using patch clamp recordings, we found that co-expression of Phactr1 with wild-type Slack channels reduces the current amplitude but has no effect on Slack channels in which a conserved PKC phosphorylation site (S407) that regulates the current amplitude has been mutated. Furthermore, a Phactr1 mutant that disrupts the binding of PP1 but not that of actin fails to alter Slack currents. Our data suggest that Phactr1 regulates the Slack by linking PP1 to the channel. Targeting Slack-Phactr1 interactions may therefore be helpful in developing the novel therapies for brain disorders associated with the malfunction of Slack channels.