The compass to follow: Focal adhesion turnover.

How cells move is a fundamental biological question. The directionality of adherent migrating cells depends on the assembly and disassembly (turnover) of focal adhesions (FAs). FAs are micron-sized actin-based structures that link cells to the extracellular matrix. Traditionally, microtubules have been considered key to triggering FA turnover. Through the years, advancements in biochemistry, biophysics, and bioimaging tools have been invaluable for many research groups to unravel a variety of mechanisms and molecular players that contribute to FA turnover, beyond microtubules. Here, we discuss recent discoveries of key molecular players that affect the dynamics and organization of the actin cytoskeleton to enable timely FA turnover and consequently proper directed cell migration.

Human septins organize as octamer-based filaments and mediate actin-membrane anchoring in cells.

Septins are cytoskeletal proteins conserved from algae and protists to mammals. A unique feature of septins is their presence as heteromeric complexes that polymerize into filaments in solution and on lipid membranes. Although animal septins associate extensively with actin-based structures in cells, whether septins organize as filaments in cells and if septin organization impacts septin function is not known. Customizing a tripartite split-GFP complementation assay, we show that all septins decorating actin stress fibers are octamer-containing filaments. Depleting octamers or preventing septins from polymerizing leads to a loss of stress fibers and reduced cell stiffness. Super-resolution microscopy revealed septin fibers with widths compatible with their organization as paired septin filaments. Nanometer-resolved distance measurements and single-protein tracking further showed that septin filaments are membrane bound and largely immobilized. Finally, reconstitution assays showed that septin filaments mediate actin-membrane anchoring. We propose that septin organization as octamer-based filaments is essential for septin function in anchoring and stabilizing actin filaments at the plasma membrane.

Bottom-Up In Vitro Methods to Assay the Ultrastructural Organization, Membrane Reshaping, and Curvature Sensitivity Behavior of Septins.

Membrane remodeling occurs constantly at the plasma membrane and within cellular organelles. To fully dissect the role of the environment (ionic conditions, protein and lipid compositions, membrane curvature) and the different partners associated with specific membrane reshaping processes, we undertake in vitro bottom-up approaches. In recent years, there has been keen interest in revealing the role of septin proteins associated with major diseases. Septins are essential and ubiquitous cytoskeletal proteins that interact with the plasma membrane. They are implicated in cell division, cell motility, neuro-morphogenesis, and spermiogenesis, among other functions. It is, therefore, important to understand how septins interact and organize at membranes to subsequently induce membrane deformations and how they can be sensitive to specific membrane curvatures. This article aims to decipher the interplay between the ultra-structure of septins at a molecular level and the membrane remodeling occurring at a micron scale. To this end, budding yeast, and mammalian septin complexes were recombinantly expressed and purified. A combination of in vitro assays was then used to analyze the self-assembly of septins at the membrane. Supported lipid bilayers (SLBs), giant unilamellar vesicles (GUVs), large unilamellar vesicles (LUVs), and wavy substrates were used to study the interplay between septin self-assembly, membrane reshaping, and membrane curvature.

Purification and Quality Control of Recombinant Septin Complexes for Cell-Free Reconstitution.

Septins are a family of conserved eukaryotic GTP-binding proteins that can form cytoskeletal filaments and higher-order structures from hetero-oligomeric complexes. They interact with other cytoskeletal components and the cell membrane to participate in important cellular functions such as migration and cell division. Due to the complexity of septins' many interactions, the large number of septin genes (13 in humans), and the ability of septins to form hetero-oligomeric complexes with different subunit compositions, cell-free reconstitution is a vital strategy to understand the basics of septin biology. The present paper first describes a method to purify recombinant septins in their hetero-oligomeric form using a two-step affinity chromatography approach. Then, the process of quality control used to check for the purity and integrity of the septin complexes is detailed. This process combines native and denaturing gel electrophoresis, negative stain electron microscopy, and interferometric scattering microscopy. Finally, a description of the process to check for the polymerization ability of septin complexes using negative stain electron microscopy and fluorescent microscopy is given. This demonstrates that it is possible to produce high-quality human septin hexamers and octamers containing different isoforms of septin_9, as well as Drosophila septin hexamers.

Septin filament compaction into rings requires the anillin Mid2 and contractile ring constriction.

Septin filaments assemble into high-order molecular structures that associate with membranes, acting as diffusion barriers and scaffold proteins crucial for many cellular processes. How septin filaments organize in such structures is still not understood. Here, we used fission yeast to explore septin filament organization during cell division and its cell cycle regulation. Live-imaging and polarization microscopy analysis uncovered that septin filaments are initially recruited as a diffuse meshwork surrounding the acto-myosin contractile ring (CR) in anaphase, which undergoes compaction into two rings when CR constriction is initiated. We found that the anillin-like protein Mid2 is necessary to promote this compaction step, possibly acting as a bundler for septin filaments. Moreover, Mid2-driven septin compaction requires inputs from the septation initiation network as well as CR constriction and the ß(1,3)-glucan synthase Bgs1. This work highlights that anillin-mediated septin ring assembly is under strict cell cycle control.

4polar-STORM polarized super-resolution imaging of actin filament organization in cells.

Single-molecule localization microscopy provides insights into the nanometer-scale spatial organization of proteins in cells, however it does not provide information on their conformation and orientation, which are key functional signatures. Detecting single molecules' orientation in addition to their localization in cells is still a challenging task, in particular in dense cell samples. Here, we present a polarization-splitting scheme which combines Stochastic Optical Reconstruction Microscopy (STORM) with single molecule 2D orientation and wobbling measurements, without requiring a strong deformation of the imaged point spread function. This method called 4polar-STORM allows, thanks to a control of its detection numerical aperture, to determine both single molecules' localization and orientation in 2D and to infer their 3D orientation. 4polar-STORM is compatible with relatively high densities of diffraction-limited spots in an image, and is thus ideally placed for the investigation of dense protein assemblies in cells. We demonstrate the potential of this method in dense actin filament organizations driving cell adhesion and motility.

Septin-microtubule association via a motif unique to isoform 1 of septin 9 tunes stress fibers.

Septins, a family of GTP-binding proteins that assemble into higher order structures, interface with the membrane, actin filaments and microtubules, and are thus important regulators of cytoarchitecture. Septin 9 (SEPT9), which is frequently overexpressed in tumors and mutated in hereditary neuralgic amyotrophy (HNA), mediates the binding of septins to microtubules, but the molecular determinants of this interaction remained uncertain. We demonstrate that a short microtubule-associated protein (MAP)-like motif unique to SEPT9 isoform 1 (SEPT9_i1) drives septin octamer-microtubule interaction in cells and in vitro reconstitutions. Septin-microtubule association requires polymerizable septin octamers harboring SEPT9_i1. Although outside of the MAP-like motif, HNA mutations abrogate this association, identifying a putative regulatory domain. Removal of this domain from SEPT9_i1 sequesters septins on microtubules, promotes microtubule stability and alters actomyosin fiber distribution and tension. Thus, we identify key molecular determinants and potential regulatory roles of septin-microtubule interaction, paving the way to deciphering the mechanisms underlying septin-associated pathologies. This article has an associated First Person interview with the first author of the paper.

Insights into animal septins using recombinant human septin octamers with distinct SEPT9 isoforms.

Septin GTP-binding proteins contribute essential biological functions that range from the establishment of cell polarity to animal tissue morphogenesis. Human septins in cells form hetero-octameric septin complexes containing the ubiquitously expressed SEPT9 subunit (also known as SEPTIN9). Despite the established role of SEPT9 in mammalian development and human pathophysiology, biochemical and biophysical studies have relied on monomeric SEPT9, thus not recapitulating its native assembly into hetero-octameric complexes. We established a protocol that enabled, for the first time, the isolation of recombinant human septin octamers containing distinct SEPT9 isoforms. A combination of biochemical and biophysical assays confirmed the octameric nature of the isolated complexes in solution. Reconstitution studies showed that octamers with either a long or a short SEPT9 isoform form filament assemblies, and can directly bind and cross-link actin filaments, raising the possibility that septin-decorated actin structures in cells reflect direct actin-septin interactions. Recombinant SEPT9-containing octamers will make it possible to design cell-free assays to dissect the complex interactions of septins with cell membranes and the actin and microtubule cytoskeleton.

Membrane binding controls ordered self-assembly of animal septins.

Septins are conserved cytoskeletal proteins that regulate cell cortex mechanics. The mechanisms of their interactions with the plasma membrane remain poorly understood. Here, we show by cell-free reconstitution that binding to flat lipid membranes requires electrostatic interactions of septins with anionic lipids and promotes the ordered self-assembly of fly septins into filamentous meshworks. Transmission electron microscopy reveals that both fly and mammalian septin hexamers form arrays of single and paired filaments. Atomic force microscopy and quartz crystal microbalance demonstrate that the fly filaments form mechanically rigid, 12- to 18-nm thick, double layers of septins. By contrast, C-terminally truncated septin mutants form 4-nm thin monolayers, indicating that stacking requires the C-terminal coiled coils on DSep2 and Pnut subunits. Our work shows that membrane binding is required for fly septins to form ordered arrays of single and paired filaments and provides new insights into the mechanisms by which septins may regulate cell surface mechanics.

Mitochondrial morphology and activity regulate furrow ingression and contractile ring dynamics in Drosophila cellularization.

Mitochondria are maternally inherited in many organisms. Mitochondrial morphology and activity regulation is essential for cell survival, differentiation, and migration. An analysis of mitochondrial dynamics and function in morphogenetic events in early metazoan embryogenesis has not been carried out. In our study we find a crucial role of mitochondrial morphology regulation in cell formation in Drosophila embryogenesis. We find that mitochondria are small and fragmented and translocate apically on microtubules and distribute progressively along the cell length during cellularization. Embryos mutant for the mitochondrial fission protein, Drp1 (dynamin-related protein 1), die in embryogenesis and show an accumulation of clustered mitochondria on the basal side in cellularization. Additionally, Drp1 mutant embryos contain lower levels of reactive oxygen species (ROS). ROS depletion was previously shown to decrease myosin II activity. Drp1 loss also leads to myosin II depletion at the membrane furrow, thereby resulting in decreased cell height and larger contractile ring area in cellularization similar to that in myosin II mutants. The mitochondrial morphology and cellularization defects in Drp1 mutants are suppressed by reducing mitochondrial fusion and increasing cytoplasmic ROS in superoxide dismutase mutants. Our data show a key role for mitochondrial morphology and activity in supporting the morphogenetic events that drive cellularization in Drosophila embryos.

The role of APC-mediated actin assembly in microtubule capture and focal adhesion turnover.

Focal adhesion (FA) turnover depends on microtubules and actin. Microtubule ends are captured at FAs, where they induce rapid FA disassembly. However, actin's roles are less clear. Here, we use polarization-resolved microscopy, FRAP, live cell imaging, and a mutant of Adenomatous polyposis coli (APC-m4) defective in actin nucleation to investigate the role of actin assembly in FA turnover. We show that APC-mediated actin assembly is critical for maintaining normal F-actin levels, organization, and dynamics at FAs, along with organization of FA components. In WT cells, microtubules are captured repeatedly at FAs as they mature, but once a FA reaches peak maturity, the next microtubule capture event leads to delivery of an autophagosome, triggering FA disassembly. In APC-m4 cells, microtubule capture frequency and duration are altered, and there are long delays between autophagosome delivery and FA disassembly. Thus, APC-mediated actin assembly is required for normal feedback between microtubules and FAs, and maintaining FAs in a state "primed" for microtubule-induced turnover.

Quantitative nanoscale imaging of orientational order in biological filaments by polarized superresolution microscopy.

Essential cellular functions as diverse as genome maintenance and tissue morphogenesis rely on the dynamic organization of filamentous assemblies. For example, the precise structural organization of DNA filaments has profound consequences on all DNA-mediated processes including gene expression, whereas control over the precise spatial arrangement of cytoskeletal protein filaments is key for mechanical force generation driving animal tissue morphogenesis. Polarized fluorescence is currently used to extract structural organization of fluorescently labeled biological filaments by determining the orientation of fluorescent labels, however with a strong drawback: polarized fluorescence imaging is indeed spatially limited by optical diffraction, and is thus unable to discriminate between the intrinsic orientational mobility of the fluorophore labels and the real structural disorder of the labeled biomolecules. Here, we demonstrate that quantitative single-molecule polarized detection in biological filament assemblies allows not only to correct for the rotational flexibility of the label but also to image orientational order of filaments at the nanoscale using superresolution capabilities. The method is based on polarized direct stochastic optical reconstruction microscopy, using dedicated optical scheme and image analysis to determine both molecular localization and orientation with high precision. We apply this method to double-stranded DNA in vitro and microtubules and actin stress fibers in whole cells.

Dynamin regulates metaphase furrow formation and plasma membrane compartmentalization in the syncytial Drosophila embryo.

The successive nuclear division cycles in the syncytial Drosophila embryo are accompanied by ingression and regression of plasma membrane furrows, which surround individual nuclei at the embryo periphery, playing a central role in embryo compartmentalization prior to cellularization. Here, we demonstrate that cell cycle changes in dynamin localization and activity at the plasma membrane (PM) regulate metaphase furrow formation and PM organization in the syncytial embryo. Dynamin was localized on short PM furrows during interphase, mediating endocytosis of PM components. Dynamin redistributed off ingressed PM furrows in metaphase, correlating with stabilized PM components and the associated actin regulatory machinery on long furrows. Acute inhibition of dynamin in the temperature sensitive shibire mutant embryo resulted in morphogenetic consequences in the syncytial division cycle. These included inhibition of metaphase furrow ingression, randomization of proteins normally polarized to intercap PM and disruption of the diffusion barrier separating PM domains above nuclei. Based on these findings, we propose that cell cycle changes in dynamin orchestrate recruitment of actin regulatory machinery for PM furrow dynamics during the early mitotic cycles in the Drosophila embryo.

Septins promote F-actin ring formation by crosslinking actin filaments into curved bundles.

Animal cell cytokinesis requires a contractile ring of crosslinked actin filaments and myosin motors. How contractile rings form and are stabilized in dividing cells remains unclear. We address this problem by focusing on septins, highly conserved proteins in eukaryotes whose precise contribution to cytokinesis remains elusive. We use the cleavage of the Drosophila melanogaster embryo as a model system, where contractile actin rings drive constriction of invaginating membranes to produce an epithelium in a manner akin to cell division. In vivo functional studies show that septins are required for generating curved and tightly packed actin filament networks. In vitro reconstitution assays show that septins alone bundle actin filaments into rings, accounting for the defects in actin ring formation in septin mutants. The bundling and bending activities are conserved for human septins, and highlight unique functions of septins in the organization of contractile actomyosin rings.

Lighting up developmental mechanisms: how fluorescence imaging heralded a new era.

Embryology and genetics have given rise to a mechanistic framework that explains the architecture of a developing organism. Until recently, however, such studies suffered from a lack of quantification and real-time visualization at the subcellular level, limiting their ability to monitor the dynamics of developmental processes. Live imaging using fluorescent proteins has overcome these limitations, uncovering unprecedented insights that call many established models into question. We review how the study of patterning, cell polarization and morphogenesis has benefited from this technology and discuss the possibilities offered by fluorescence imaging and by the contributions of quantitative disciplines.

Cells within a cell: Insights into cellular architecture and polarization from the organization of the early fly embryo.

Drosophila embryogenesis begins with 13 rapid nuclear divisions within a common cytoplasm. These divisions produce approximately 6,000 nuclei that, during the next division cycle, become encased in plasma membrane (PM) and generate the primary embryonic epithelium in the process known as cellularization. Despite the absence of PM boundaries between syncytial nuclei, the secretory membrane system is organized in functionally compartmentalized units around individual nuclei.1 We have recently used in vivo fluorescence imaging to characterize the dynamics of proteins in the PM of the embryonic syncytium. These studies revealed that the PM is polarized already before cellularization. One PM region resides above individual nuclei and has apical-like features, while PM regions lateral to nuclei have basolateral characteristics. Optical highlighting experiments showed that membrane components do not exchange between PM regions that reside above adjacent nuclei. An intact F-actin network was shown to be important for both the PM apicobasallike polarity and the diffusion barriers within the syncytial PM. Our findings, as well as their possible implications, are further discussed in this Addendum.

Plasma membrane polarity and compartmentalization are established before cellularization in the fly embryo.

Patterning in the Drosophila embryo requires local activation and dynamics of proteins in the plasma membrane (PM). We used in vivo fluorescence imaging to characterize the organization and diffusional properties of the PM in the early embryonic syncytium. Before cellularization, the PM is polarized into discrete domains having epithelial-like characteristics. One domain resides above individual nuclei and has apical-like characteristics, while the other domain is lateral to nuclei and contains markers associated with basolateral membranes and junctions. Pulse-chase photoconversion experiments show that molecules can diffuse within each domain but do not exchange between PM regions above adjacent nuclei. Drug-induced F-actin depolymerization disrupted both the apicobasal-like polarity and the diffusion barriers within the syncytial PM. These events correlated with perturbations in the spatial pattern of dorsoventral Toll signaling. We propose that epithelial-like properties and an intact F-actin network compartmentalize the PM and shape morphogen gradients in the syncytial embryo.

Fluorescence imaging techniques for studying Drosophila embryo development.

This unit describes fluorescence-based techniques for noninvasive imaging of development in living Drosophila embryos, discussing considerations for fluorescent imaging within living embryos and providing protocols for generation of flies expressing fluorescently tagged proteins and for preparation of embryos for fluorescent imaging. The unit details time-lapse confocal imaging of live embryos and discusses optimizing image acquisition and performing three-dimensional imaging. Finally, the unit provides a variety of specific methods for optical highlighting of specific subsets of fluorescently tagged proteins and organelles in the embryo, including fluorescence recovery after photobleaching (FRAP), fluorescence loss in photobleaching (FLIP), and photoactivation techniques, permitting analysis of specific movements of fluorescently tagged proteins within cells. These protocols, together with the relative ease of generating transgenic animals and the ability to express tagged proteins in specific tissues or at specific developmental times, provide powerful means for examining in vivo behavior of any tagged protein in embryos in myriad mutant backgrounds.

Protein kinase A effects of an expressed PRKAR1A mutation associated with aggressive tumors.

Most PRKAR1A tumorigenic mutations lead to nonsense mRNA that is decayed; tumor formation has been associated with an increase in type II protein kinase A (PKA) subunits. The IVS6+1G>T PRKAR1A mutation leads to a protein lacking exon 6 sequences [R1 alpha Delta 184-236 (R1 alpha Delta 6)]. We compared in vitro R1 alpha Delta 6 with wild-type (wt) R1 alpha. We assessed PKA activity and subunit expression, phosphorylation of target molecules, and properties of wt-R1 alpha and mutant (mt) R1 alpha; we observed by confocal microscopy R1 alpha tagged with green fluorescent protein and its interactions with Cerulean-tagged catalytic subunit (C alpha). Introduction of the R1 alpha Delta 6 led to aberrant cellular morphology and higher PKA activity but no increase in type II PKA subunits. There was diffuse, cytoplasmic localization of R1 alpha protein in wt-R1 alpha- and R1 alpha Delta 6-transfected cells but the former also exhibited discrete aggregates of R1 alpha that bound C alpha; these were absent in R1 alpha Delta 6-transfected cells and did not bind C alpha at baseline or in response to cyclic AMP. Other changes induced by R1 alpha Delta 6 included decreased nuclear C alpha. We conclude that R1 alpha Delta 6 leads to increased PKA activity through the mt-R1 alpha decreased binding to C alpha and does not involve changes in other PKA subunits, suggesting that a switch to type II PKA activity is not necessary for increased kinase activity or tumorigenesis.

Homeostatic mechanisms for iron storage revealed by genetic manipulations and live imaging of Drosophila ferritin.

Ferritin is a symmetric, 24-subunit iron-storage complex assembled of H and L chains. It is found in bacteria, plants, and animals and in two classes of mutations in the human L-chain gene, resulting in hereditary hyperferritinemia cataract syndrome or in neuroferritinopathy. Here, we examined systemic and cellular ferritin regulation and trafficking in the model organism Drosophila melanogaster. We showed that ferritin H and L transcripts are coexpressed during embryogenesis and that both subunits are essential for embryonic development. Ferritin overexpression impaired the survival of iron-deprived flies. In vivo expression of GFP-tagged holoferritin confirmed that iron-loaded ferritin molecules traffic through the Golgi organelle and are secreted into hemolymph. A constant ratio of ferritin H and L subunits, secured via tight post-transcriptional regulation, is characteristic of the secreted ferritin in flies. Differential cellular expression, conserved post-transcriptional regulation via the iron regulatory element, and distinct subcellular localization of the ferritin subunits prior to the assembly of holoferritin are all important steps mediating iron homeostasis. Our study revealed both conserved features and insect-specific adaptations of ferritin nanocages and provides novel imaging possibilities for their in vivo characterization.