Binding partners regulate unfolding of myosin VI to activate the molecular motor.

Myosin VI is the only minus-end actin motor and it is coupled to various cellular processes ranging from endocytosis to transcription. This multi-potent nature is achieved through alternative isoform splicing and interactions with a network of binding partners. There is a complex interplay between isoforms and binding partners to regulate myosin VI. Here, we have compared the regulation of two myosin VI splice isoforms by two different binding partners. By combining biochemical and single-molecule approaches, we propose that myosin VI regulation follows a generic mechanism, independently of the spliced isoform and the binding partner involved. We describe how myosin VI adopts an autoinhibited backfolded state which is released by binding partners. This unfolding activates the motor, enhances actin binding and can subsequently trigger dimerization. We have further expanded our study by using single-molecule imaging to investigate the impact of binding partners upon myosin VI molecular organization and dynamics.

DNA damage alters nuclear mechanics through chromatin reorganization.

DNA double-strand breaks drive genomic instability. However, it remains unknown how these processes may affect the biomechanical properties of the nucleus and what role nuclear mechanics play in DNA damage and repair efficiency. Here, we have used Atomic Force Microscopy to investigate nuclear mechanical changes, arising from externally induced DNA damage. We found that nuclear stiffness is significantly reduced after cisplatin treatment, as a consequence of DNA damage signalling. This softening was linked to global chromatin decondensation, which improves molecular diffusion within the organelle. We propose that this can increase recruitment for repair factors. Interestingly, we also found that reduction of nuclear tension, through cytoskeletal relaxation, has a protective role to the cell and reduces accumulation of DNA damage. Overall, these changes protect against further genomic instability and promote DNA repair. We propose that these processes may underpin the development of drug resistance.

Talin mechanosensitivity is modulated by a direct interaction with cyclin-dependent kinase-1.

Talin (TLN1) is a mechanosensitive component of adhesion complexes that directly couples integrins to the actin cytoskeleton. In response to force, talin undergoes switch-like behavior of its multiple rod domains that modulate interactions with its binding partners. Cyclin-dependent kinase-1 (CDK1) is a key regulator of the cell cycle, exerting its effects through synchronized phosphorylation of a large number of protein targets. CDK1 activity maintains adhesion during interphase, and its inhibition is a prerequisite for the tightly choreographed changes in cell shape and adhesion that are required for successful mitosis. Using a combination of biochemical, structural, and cell biological approaches, we demonstrate a direct interaction between talin and CDK1 that occurs at sites of integrin-mediated adhesion. Mutagenesis demonstrated that CDK1 contains a functional talin-binding LD motif, and the binding site within talin was pinpointed to helical bundle R8. Talin also contains a consensus CDK1 phosphorylation motif centered on S1589, a site shown to be phosphorylated by CDK1 in vitro. A phosphomimetic mutant of this site within talin lowered the binding affinity of the cytoskeletal adaptor KANK and weakened the response of this region to force as measured by single molecule stretching, potentially altering downstream mechanotransduction pathways. The direct binding of the master cell cycle regulator CDK1 to the primary integrin effector talin represents a coupling of cell proliferation and cell adhesion machineries and thereby indicates a mechanism by which the microenvironment can control cell division in multicellular organisms.

Nuclear myosins - roles for molecular transporters and anchors.

The myosin family of molecular motors are well-characterised cytoskeletal proteins. However, myosins are also present in the nucleus, where they have been shown to have roles in transcription, DNA repair and viral infections. Despite their involvement in these fundamental cellular processes, our understanding of these functions and their regulation remains limited. Recently, research on nuclear myosins has been gathering pace, and this Review will evaluate the current state of the field. Here, we will focus on the variation in structure of nuclear myosins, their nuclear import and their roles within transcription, DNA damage, chromatin organisation and viral infections. We will also consider both the biochemical and biophysical properties and restraints that are placed on these multifunctional motors, and how they link to their cytoplasmic counterparts. By highlighting these properties and processes, we show just how integral nuclear myosins are for cellular survival.

Competition between two high- and low-affinity protein-binding sites in myosin VI controls its cellular function.

Myosin VI is involved in many cellular processes ranging from endocytosis to transcription. This multifunctional potential is achieved through alternative isoform splicing and through interactions of myosin VI with a diverse network of binding partners. However, the interplay between these two modes of regulation remains unexplored. To this end, we compared two different binding partners and their interactions with myosin VI by exploring the kinetic properties of recombinant proteins and their distribution in mammalian cells using fluorescence imaging. We found that selectivity for these binding partners is achieved through a high-affinity motif and a low-affinity motif within myosin VI. These two motifs allow competition among partners for myosin VI. Exploring how this competition affects the activity of nuclear myosin VI, we demonstrate the impact of a concentration-driven interaction with the low-affinity binding partner DAB2, finding that this interaction blocks the ability of nuclear myosin VI to bind DNA and its transcriptional activity in vitro We conclude that loss of DAB2, a tumor suppressor, may enhance myosin VI-mediated transcription. We propose that the frequent loss of specific myosin VI partner proteins during the onset of cancer leads to a higher level of nuclear myosin VI activity.

Force-Dependent Interactions between Talin and Full-Length Vinculin.

Talin and vinculin are part of a multicomponent system involved in mechanosensing in cell-matrix adhesions. Both exist in autoinhibited forms, and activation of vinculin requires binding to mechanically activated talin, yet how forces affect talin's interaction with vinculin has not been investigated. Here by quantifying the kinetics of force-dependent talin-vinculin interactions using single-molecule analysis, we show that mechanical exposure of a single vinculin binding site (VBS) in talin is sufficient to relieve the autoinhibition of vinculin, resulting in high-affinity binding. We provide evidence that the vinculin undergoes dynamic fluctuations between an autoinhibited closed conformation and an open conformation that is stabilized upon binding to the VBS. Furthermore, we discover an additional level of regulation in which the mechanically exposed VBS binds vinculin significantly more tightly than the isolated VBS alone. Molecular dynamics simulations reveal the basis of this new regulatory mechanism, identifying a sensitive force-dependent change in the conformation of an exposed VBS that modulates binding. Together, these results provide a comprehensive understanding of how the interplay between force and autoinhibition provides exquisite complexity within this major mechanosensing axis.

Proteomic profiling identifies a direct interaction between heat shock transcription factor 2 and the focal adhesion adapter talin-1.

Heat shock factor 2 (HSF2) is a versatile transcription factor that regulates gene expression under stress conditions, during development, and in disease. Despite recent advances in characterizing HSF2-dependent target genes, little is known about the protein networks associated with this transcription factor. In this study, we performed co-immunoprecipitation coupled with mass spectrometry analysis to identify the HSF2 interactome in mouse testes, where HSF2 is required for normal sperm development. Endogenous HSF2 was discovered to form a complex with several adhesion-associated proteins, a finding substantiated by mass spectrometry analysis conducted in human prostate carcinoma PC-3 cells. Notably, this group of proteins included the focal adhesion adapter protein talin-1 (TLN1). Through co-immunoprecipitation and proximity ligation assays, we demonstrate the conservation of the HSF2-TLN1 interaction from mouse to human. Additionally, employing sequence alignment analyses, we uncovered a TLN1-binding motif in the HSF2 C terminus that binds directly to multiple regions of TLN1 in vitro. We provide evidence that the 25 C-terminal amino acids of HSF2, fused to EGFP, are sufficient to establish a protein complex with TLN1 and modify cell-cell adhesion in human cells. Importantly, this TLN1-binding motif is absent in the C-terminus of a closely related HSF family member, HSF1, which does not form a complex with TLN1. These results highlight the unique molecular characteristics of HSF2 in comparison to HSF1. Taken together, our data unveil the protein partners associated with HSF2 in a physiologically relevant context and identifies TLN1 as the first adhesion-related HSF2-interacting partner.

Use of Online Health Forums by People Living With Breast Cancer During the COVID-19 Pandemic: Thematic Analysis.

BACKGROUND: At the time of the UK COVID-19 lockdowns, online health forums (OHFs) were one of the relatively few remaining accessible sources of peer support for people living with breast cancer. Cancer services were heavily affected by the pandemic in many ways, including the closure of many of the customary support services. Previous studies indicate that loneliness, anxiety, distress, and depression caused by COVID-19 were common among people living with breast cancer, and this suggests that the role of OHFs in providing users with support, information, and empathy could have been of increased importance at that time. OBJECTIVE: This study aimed to examine how people living with breast cancer shared information, experiences, and emotions in an OHF during the COVID-19 pandemic. METHODS: This qualitative study thematically analyzed posts from the discussion forums of an OHF provided by the UK charity, Breast Cancer Now. We selected 1053 posts from the time of 2 UK lockdowns: March 16, 2020, to June 15, 2020 (lockdown 1), and January 6, 2021, to March 8, 2021 (lockdown 3), for analysis, from 2 of the forum's boards (for recently diagnosed people and for those undergoing chemotherapy). We analyzed the data using the original 6 steps for thematic analysis by Braun and Clarke but by following a codebook approach. Descriptive statistics for posts were also derived. RESULTS: We found that COVID-19 amplified the forum's value to its users. As patients with cancer, participants were in a situation that was "bad enough already," and the COVID-19 pandemic heightened this difficult situation. The forum's value, which was already high for the information and peer support it provided, increased because COVID-19 caused some special information needs that forum users were uniquely well placed to fulfill as people experiencing the combined effects of having breast cancer during the pandemic. The forum also met the emotional needs generated by the COVID-19 pandemic and was valued as a place where loneliness during the pandemic may be relieved and users' spirits lifted in a variety of ways specific to this period. We found some differences in use between the 2 periods and the 2 boards-most noticeable was the great fear and anxiety expressed at the beginning of lockdown 1. Both the beginning and end of lockdown periods were particularly difficult for participants, with the ends seen as potentially increasing isolation. CONCLUSIONS: The forums were an important source of support and information to their users, with their value increasing during the lockdowns for a variety of reasons. Our findings will be helpful to organizations offering OHFs and to health care workers advising people living with breast cancer about sources of support.

Measuring Nuclear Organization of Proteins with STORM Imaging and Cluster Analysis.

Super-resolution microscopy enables the high-precision localization of proteins. Therefore, it is possible to investigate the spatial organization of proteins within the nucleus to understand how their organization relates to regulation and function. Here, we present methodology for single-molecule localization microscopy and cluster analysis where we cover sample preparation, image acquisition, and data analysis.

Myosin VI regulates the spatial organisation of mammalian transcription initiation.

During transcription, RNA Polymerase II (RNAPII) is spatially organised within the nucleus into clusters that correlate with transcription activity. While this is a hallmark of genome regulation in mammalian cells, the mechanisms concerning the assembly, organisation and stability remain unknown. Here, we have used combination of single molecule imaging and genomic approaches to explore the role of nuclear myosin VI (MVI) in the nanoscale organisation of RNAPII. We reveal that MVI in the nucleus acts as the molecular anchor that holds RNAPII in high density clusters. Perturbation of MVI leads to the disruption of RNAPII localisation, chromatin organisation and subsequently a decrease in gene expression. Overall, we uncover the fundamental role of MVI in the spatial regulation of gene expression.

The tale of two talins - two isoforms to fine-tune integrin signalling.

Talins are cytoplasmic adapter proteins essential for integrin-mediated cell adhesion to the extracellular matrix. Talins control the activation state of integrins, link integrins to cytoskeletal actin, recruit numerous signalling molecules that mediate integrin signalling and coordinate recruitment of microtubules to adhesion sites via interaction with KANK (kidney ankyrin repeat-containing) proteins. Vertebrates have two talin genes, TLN1 and TLN2. Although talin1 and talin2 share 76% protein sequence identity (88% similarity), they are not functionally redundant, and the differences between the two isoforms are not fully understood. In this Review, we focus on the similarities and differences between the two talins in terms of structure, biochemistry and function, which hint at subtle differences in fine-tuning adhesion signalling.

Talin-KANK1 interaction controls the recruitment of cortical microtubule stabilizing complexes to focal adhesions.

The cross-talk between dynamic microtubules and integrin-based adhesions to the extracellular matrix plays a crucial role in cell polarity and migration. Microtubules regulate the turnover of adhesion sites, and, in turn, focal adhesions promote the cortical microtubule capture and stabilization in their vicinity, but the underlying mechanism is unknown. Here, we show that cortical microtubule stabilization sites containing CLASPs, KIF21A, LL5ß and liprins are recruited to focal adhesions by the adaptor protein KANK1, which directly interacts with the major adhesion component, talin. Structural studies showed that the conserved KN domain in KANK1 binds to the talin rod domain R7. Perturbation of this interaction, including a single point mutation in talin, which disrupts KANK1 binding but not the talin function in adhesion, abrogates the association of microtubule-stabilizing complexes with focal adhesions. We propose that the talin-KANK1 interaction links the two macromolecular assemblies that control cortical attachment of actin fibers and microtubules.