Identification of F-actin as the dynamic hub in a microbial-induced GTPase polarity circuit.

Polarity in mammalian cells emerges from the assembly of signaling molecules into extensive biochemical interaction networks. Despite their complexity, bacterial pathogens have evolved parsimonious mechanisms to hijack these systems. Here, we develop a tractable experimental and theoretical model to uncover fundamental operating principles, in both mammalian cell polarity and bacterial pathogenesis. Using synthetic derivatives of the enteropathogenic Escherichia coli guanine-nucleotide exchange factor (GEF) Map, we discover that Cdc42 GTPase signal transduction is controlled by the interaction between Map and F-actin. Mathematical modeling reveals how actin dynamics coupled to a Map-dependent positive feedback loop spontaneously polarizes Cdc42 on the plasma membrane. By rewiring the pathogenic signaling circuit to operate through ß-integrin stimulation, we further show how Cdc42 is polarized in response to an extracellular spatial cue. Thus, a molecular pathway of polarity is proposed, centered on the interaction between GEFs and F-actin, which is likely to function in diverse biological systems.

Nuclear lamin isoforms differentially contribute to LINC complex-dependent nucleocytoskeletal coupling and whole-cell mechanics.

The ability of a cell to regulate its mechanical properties is central to its function. Emerging evidence suggests that interactions between the cell nucleus and cytoskeleton influence cell mechanics through poorly understood mechanisms. Here we conduct quantitative confocal imaging to show that the loss of A-type lamins tends to increase nuclear and cellular volume while the loss of B-type lamins behaves in the opposite manner. We use fluorescence recovery after photobleaching, atomic force microscopy, optical tweezer microrheology, and traction force microscopy to demonstrate that A-type lamins engage with both F-actin and vimentin intermediate filaments (VIFs) through the linker of nucleoskeleton and cytoskeleton (LINC) complexes to modulate cortical and cytoplasmic stiffness as well as cellular contractility in mouse embryonic fibroblasts (MEFs). In contrast, we show that B-type lamins predominantly interact with VIFs through LINC complexes to regulate cytoplasmic stiffness and contractility. We then propose a physical model mediated by the lamin­LINC complex that explains these distinct mechanical phenotypes (mechanophenotypes). To verify this model, we use dominant negative constructs and RNA interference to disrupt the LINC complexes that facilitate the interaction of the nucleus with the F-actin and VIF cytoskeletons and show that the loss of these elements results in mechanophenotypes like those observed in MEFs that lack A- or B-type lamin isoforms. Finally, we demonstrate that the loss of each lamin isoform softens the cell nucleus and enhances constricted cell migration but in turn increases migration-induced DNA damage. Together, our findings uncover distinctive roles for each of the four major lamin isoforms in maintaining nucleocytoskeletal interactions and cellular mechanics.

Gigaxonin is required for intermediate filament transport.

Gigaxonin is an adaptor protein for E3 ubiquitin ligase substrates. It is necessary for ubiquitination and degradation of intermediate filament (IF) proteins. Giant axonal neuropathy is a pathological condition caused by mutations in the GAN gene that encodes gigaxonin. This condition is characterized by abnormal accumulation of IFs in both neuronal and non-neuronal cells; however, it is unclear what causes IF aggregation. In this work, we studied the dynamics of IFs using their subunits tagged with a photoconvertible protein mEOS 3.2. We have demonstrated that the loss of gigaxonin dramatically inhibited transport of IFs along microtubules by the microtubule motor kinesin-1. This inhibition was specific for IFs, as other kinesin-1 cargoes, with the exception of mitochondria, were transported normally. Abnormal distribution of IFs in the cytoplasm can be rescued by direct binding of kinesin-1 to IFs, demonstrating that transport inhibition is the primary cause for the abnormal IF distribution. Another effect of gigaxonin loss was a more than 20-fold increase in the amount of soluble vimentin oligomers in the cytosol of gigaxonin knock-out cells. We speculate that these oligomers saturate a yet unidentified adapter that is required for kinesin-1 binding to IFs, which might inhibit IF transport along microtubules causing their abnormal accumulation.

OME-Zarr: a cloud-optimized bioimaging file format with international community support.

A growing community is constructing a next-generation file format (NGFF) for bioimaging to overcome problems of scalability and heterogeneity. Organized by the Open Microscopy Environment (OME), individuals and institutes across diverse modalities facing these problems have designed a format specification process (OME-NGFF) to address these needs. This paper brings together a wide range of those community members to describe the cloud-optimized format itself-OME-Zarr-along with tools and data resources available today to increase FAIR access and remove barriers in the scientific process. The current momentum offers an opportunity to unify a key component of the bioimaging domain-the file format that underlies so many personal, institutional, and global data management and analysis tasks.

NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and nuclear envelope rupture.

Neutrophil extracellular traps (NETs) are web-like DNA structures decorated with histones and cytotoxic proteins that are released by activated neutrophils to trap and neutralize pathogens during the innate immune response, but also form in and exacerbate sterile inflammation. Peptidylarginine deiminase 4 (PAD4) citrullinates histones and is required for NET formation (NETosis) in mouse neutrophils. While the in vivo impact of NETs is accumulating, the cellular events driving NETosis and the role of PAD4 in these events are unclear. We performed high-resolution time-lapse microscopy of mouse and human neutrophils and differentiated HL-60 neutrophil-like cells (dHL-60) labeled with fluorescent markers of organelles and stimulated with bacterial toxins or Candida albicans to induce NETosis. Upon stimulation, cells exhibited rapid disassembly of the actin cytoskeleton, followed by shedding of plasma membrane microvesicles, disassembly and remodeling of the microtubule and vimentin cytoskeletons, ER vesiculation, chromatin decondensation and nuclear rounding, progressive plasma membrane and nuclear envelope (NE) permeabilization, nuclear lamin meshwork and then NE rupture to release DNA into the cytoplasm, and finally plasma membrane rupture and discharge of extracellular DNA. Inhibition of actin disassembly blocked NET release. Mouse and dHL-60 cells bearing genetic alteration of PAD4 showed that chromatin decondensation, lamin meshwork and NE rupture and extracellular DNA release required the enzymatic and nuclear localization activities of PAD4. Thus, NETosis proceeds by a stepwise sequence of cellular events culminating in the PAD4-mediated expulsion of DNA.

Universal light-sheet generation with field synthesis.

We introduce field synthesis, a theorem and method that can be used to synthesize any scanned or dithered light sheet, including those used in lattice light-sheet microscopy (LLSM), from an incoherent superposition of one-dimensional intensity distributions. Compared to LLSM, this user-friendly and modular approach offers a simplified optical design, higher light throughput and simultaneous multicolor illumination. Further, field synthesis achieves lower rates of photobleaching than light sheets generated by lateral beam scanning.

Adaptive multiorientation resolution analysis of complex filamentous network images.

MOTIVATION: Microscopy images of cytoskeletal, nucleoskeletal and other structures contain complex junctions of overlapping filaments with arbitrary geometry. Yet, state-of-the-art algorithms generally perform single orientation analysis to segment these structures, resulting in gaps near junctions, or assume particular junction geometries to detect them. RESULTS: We developed a fully automated image analysis approach to address the challenge of determining the number of orientations and their values at each point in space to detect both lines and their junctions. Our approach does not assume any fixed number of orientations or any particular geometry in the case of multiple coincident orientations. It is based on analytically resolving coincident orientations revealed by steerable ridge filtering in an adaptive manner that balances orientation resolution and spatial localization. Combining this multiorientation resolution information with a generalization of the concept of non-maximum suppression allowed us to then identify the centers of lines and their junctions in an image. We validated our approach using a wide array of synthetic junctions and by comparison to manual segmentation. We also applied it to light microscopy images of cytoskeletal and nucleoskeletal networks. AVAILABILITY AND IMPLEMENTATION: https://github.com/mkitti/AdaptiveResolutionOrientationSpace. SUPPLEMENTARY INFORMATION: Supplementary information is available at Bioinformatics online.

Kinesin-dependent transport of keratin filaments: a unified mechanism for intermediate filament transport.

Keratin intermediate filaments (IFs) are the major cytoskeletal component in epithelial cells. The dynamics of keratin IFs have been described to depend mostly on the actin cytoskeleton, but the rapid transport of fully polymerized keratin filaments has not been reported. In this work, we used a combination of photoconversion experiments and clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 genome editing to study the role of microtubules and microtubule motors in keratin filament transport. We found that long keratin filaments, like other types of IFs, are transported along microtubules by kinesin-1. Our data revealed that keratin and vimentin are nonconventional kinesin-1 cargoes because their transport did not require kinesin light chains, which are a typical adapter for kinesin-dependent cargo transport. Furthermore, we found that the same domain of the kinesin heavy chain tail is involved in keratin and vimentin IF transport, strongly suggesting that multiple types of IFs move along microtubules using an identical mechanism.-Robert, A., Tian, P., Adam, S. A., Kittisopikul, M., Jaqaman, K., Goldman, R. D., Gelfand, V. I. Kinesin-dependent transport of keratin filaments: a unified mechanism for intermediate filament transport.

Noise Expands the Response Range of the Bacillus subtilis Competence Circuit.

Gene regulatory circuits must contend with intrinsic noise that arises due to finite numbers of proteins. While some circuits act to reduce this noise, others appear to exploit it. A striking example is the competence circuit in Bacillus subtilis, which exhibits much larger noise in the duration of its competence events than a synthetically constructed analog that performs the same function. Here, using stochastic modeling and fluorescence microscopy, we show that this larger noise allows cells to exit terminal phenotypic states, which expands the range of stress levels to which cells are responsive and leads to phenotypic heterogeneity at the population level. This is an important example of how noise confers a functional benefit in a genetic decision-making circuit.

Localized cell death focuses mechanical forces during 3D patterning in a biofilm.

From microbial biofilm communities to multicellular organisms, 3D macroscopic structures develop through poorly understood interplay between cellular processes and mechanical forces. Investigating wrinkled biofilms of Bacillus subtilis, we discovered a pattern of localized cell death that spatially focuses mechanical forces, and thereby initiates wrinkle formation. Deletion of genes implicated in biofilm development, together with mathematical modeling, revealed that ECM production underlies the localization of cell death. Simultaneously with cell death, we quantitatively measured mechanical stiffness and movement in WT and mutant biofilms. Results suggest that localized cell death provides an outlet for lateral compressive forces, thereby promoting vertical mechanical buckling, which subsequently leads to wrinkle formation. Guided by these findings, we were able to generate artificial wrinkle patterns within biofilms. Formation of 3D structures facilitated by cell death may underlie self-organization in other developmental systems, and could enable engineering of macroscopic structures from cell populations.

Biological role of noise encoded in a genetic network motif.

Genetic circuits that regulate distinct cellular processes can differ in their wiring pattern of interactions (architecture) and susceptibility to stochastic fluctuations (noise). Whether the link between circuit architecture and noise is of biological importance remains, however, poorly understood. To investigate this problem, we performed a computational study of gene expression noise for all possible circuit architectures of feed-forward loop (FFL) motifs. Results revealed that FFL architectures fall into two categories depending on whether their ON (stimulated) or OFF (unstimulated) steady states exhibit noise. To explore the biological importance of this difference in noise behavior, we analyzed 858 documented FFLs in Escherichia coli that were divided into 39 functional categories. The majority of FFLs were found to regulate two subsets of functional categories. Interestingly, these two functional categories associated with FFLs of opposite noise behaviors. This opposite noise preference revealed two noise-based strategies to cope with environmental constraints where cellular responses are either initiated or terminated stochastically to allow probabilistic sampling of alternative states. FFLs may thus be selected for their architecture-dependent noise behavior, revealing a biological role for noise that is encoded in gene circuit architectures.

OME-Zarr: a cloud-optimized bioimaging file format with international community support.

A growing community is constructing a next-generation file format (NGFF) for bioimaging to overcome problems of scalability and heterogeneity. Organized by the Open Microscopy Environment (OME), individuals and institutes across diverse modalities facing these problems have designed a format specification process (OME-NGFF) to address these needs. This paper brings together a wide range of those community members to describe the cloud-optimized format itself -- OME-Zarr -- along with tools and data resources available today to increase FAIR access and remove barriers in the scientific process. The current momentum offers an opportunity to unify a key component of the bioimaging domain -- the file format that underlies so many personal, institutional, and global data management and analysis tasks.

Computational analyses reveal spatial relationships between nuclear pore complexes and specific lamins.

Nuclear lamin isoforms form fibrous meshworks associated with nuclear pore complexes (NPCs). Using datasets prepared from subpixel and segmentation analyses of 3D-structured illumination microscopy images of WT and lamin isoform knockout mouse embryo fibroblasts, we determined with high precision the spatial association of NPCs with specific lamin isoform fibers. These relationships are retained in the enlarged lamin meshworks of Lmna-/- and Lmnb1-/- fibroblast nuclei. Cryo-ET observations reveal that the lamin filaments composing the fibers contact the nucleoplasmic ring of NPCs. Knockdown of the ring-associated nucleoporin ELYS induces NPC clusters that exclude lamin A/C fibers but include LB1 and LB2 fibers. Knockdown of the nucleoporin TPR or NUP153 alters the arrangement of lamin fibers and NPCs. Evidence that the number of NPCs is regulated by specific lamin isoforms is presented. Overall the results demonstrate that lamin isoforms and nucleoporins act together to maintain the normal organization of lamin meshworks and NPCs within the nuclear envelope.

Heterogeneity in VEGF Receptor-2 Mobility and Organization on the Endothelial Cell Surface Leads to Diverse Models of Activation by VEGF.

The dynamic nanoscale organization of cell surface receptors plays an important role in signaling. We determine this organization and its relation to activation of VEGF receptor-2 (VEGFR-2), a critical receptor tyrosine kinase in endothelial cells (ECs), by combining single-molecule imaging of endogenous VEGFR-2 in live ECs with multiscale computational analysis. We find that surface VEGFR-2 can be mobile or exhibit restricted mobility and be monomeric or non-monomeric, with a complex interplay between the two. This basal heterogeneity results in heterogeneity in the sequence of steps leading to VEGFR-2 activation by VEGF. Specifically, we find that VEGF can bind to monomeric and non-monomeric VEGFR-2 and that, when binding to monomeric VEGFR-2, its effect on dimerization depends on the mobility of VEGFR-2. Our study highlights the dynamic and heterogeneous nature of cell surface receptor organization and the need for multiscale, single-molecule-based analysis to determine its relationship to receptor activation and signaling.

Community standards for open cell migration data.

Cell migration research has become a high-content field. However, the quantitative information encapsulated in these complex and high-dimensional datasets is not fully exploited owing to the diversity of experimental protocols and non-standardized output formats. In addition, typically the datasets are not open for reuse. Making the data open and Findable, Accessible, Interoperable, and Reusable (FAIR) will enable meta-analysis, data integration, and data mining. Standardized data formats and controlled vocabularies are essential for building a suitable infrastructure for that purpose but are not available in the cell migration domain. We here present standardization efforts by the Cell Migration Standardisation Organisation (CMSO), an open community-driven organization to facilitate the development of standards for cell migration data. This work will foster the development of improved algorithms and tools and enable secondary analysis of public datasets, ultimately unlocking new knowledge of the complex biological process of cell migration.

Quantitative Analysis of Nuclear Lamins Imaged by Super-Resolution Light Microscopy.

The nuclear lamina consists of a dense fibrous meshwork of nuclear lamins, Type V intermediate filaments, and is ~14 nm thick according to recent cryo-electron tomography studies. Recent advances in light microscopy have extended the resolution to a scale allowing for the fine structure of the lamina to be imaged in the context of the whole nucleus. We review quantitative approaches to analyze the imaging data of the nuclear lamina as acquired by structured illumination microscopy (SIM) and single molecule localization microscopy (SMLM), as well as the requisite cell preparation techniques. In particular, we discuss the application of steerable filters and graph-based methods to segment the structure of the four mammalian lamin isoforms (A, C, B1, and B2) and extract quantitative information.

Vimentin protects cells against nuclear rupture and DNA damage during migration.

Mammalian cells frequently migrate through tight spaces during normal embryogenesis, wound healing, diapedesis, or in pathological situations such as metastasis. Nuclear size and shape are important factors in regulating the mechanical properties of cells during their migration through such tight spaces. At the onset of migratory behavior, cells often initiate the expression of vimentin, an intermediate filament protein that polymerizes into networks extending from a juxtanuclear cage to the cell periphery. However, the role of vimentin intermediate filaments (VIFs) in regulating nuclear shape and mechanics remains unknown. Here, we use wild-type and vimentin-null mouse embryonic fibroblasts to show that VIFs regulate nuclear shape and perinuclear stiffness, cell motility in 3D, and the ability of cells to resist large deformations. These changes increase nuclear rupture and activation of DNA damage repair mechanisms, which are rescued by exogenous reexpression of vimentin. Our findings show that VIFs provide mechanical support to protect the nucleus and genome during migration.

Structural organization of nuclear lamins A, C, B1, and B2 revealed by superresolution microscopy.

The nuclear lamina is a key structural element of the metazoan nucleus. However, the structural organization of the major proteins composing the lamina is poorly defined. Using three-dimensional structured illumination microscopy and computational image analysis, we characterized the supramolecular structures of lamin A, C, B1, and B2 in mouse embryo fibroblast nuclei. Each isoform forms a distinct fiber meshwork, with comparable physical characteristics with respect to mesh edge length, mesh face area and shape, and edge connectivity to form faces. Some differences were found in face areas among isoforms due to variation in the edge lengths and number of edges per face, suggesting that each meshwork has somewhat unique assembly characteristics. In fibroblasts null for the expression of either lamins A/C or lamin B1, the remaining lamin meshworks are altered compared with the lamin meshworks in wild-type nuclei or nuclei lacking lamin B2. Nuclei lacking LA/C exhibit slightly enlarged meshwork faces and some shape changes, whereas LB1-deficient nuclei exhibit primarily a substantial increase in face area. These studies demonstrate that individual lamin isoforms assemble into complex networks within the nuclear lamina and that A- and B-type lamins have distinct roles in maintaining the organization of the nuclear lamina.