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We designed an epi-illumination SPIM system that uses a single objective and has a sample interface identical to that of an inverted fluorescence microscope with no additional reflection elements. It achieves subcellular resolution and single-molecule sensitivity, and is compatible with common biological sample holders, including multi-well plates. We demonstrated multicolor fast volumetric imaging, single-molecule localization microscopy, parallel imaging of 16 cell lines and parallel recording of cellular responses to perturbations.Epi-illumination SPIM enables fast, volumetric, high-resolution, subcellular imaging of any sample compatible with a standard inverted fluorescence microscope.

Actin filaments assemble inside the nucleus in response to multiple cellular perturbations, including heat shock, protein misfolding, integrin engagement, and serum stimulation. We find that DNA damage also generates nuclear actin filaments—detectable by phalloidin and live-cell actin probes—with three characteristic morphologies: (i) long, nucleoplasmic filaments; (ii) short, nucleolus-associated filaments; and (iii) dense, nucleoplasmic clusters. This DNA damage-induced nuclear actin assembly requires two biologically and physically linked nucleation factors: Formin-2 and Spire-1/Spire-2. Formin-2 accumulates in the nucleus after DNA damage, and depletion of either Formin-2 or actin's nuclear import factor, importin-9, increases the number of DNA double-strand breaks (DSBs), linking nuclear actin filaments to efficient DSB clearance. Nuclear actin filaments are also required for nuclear oxidation induced by acute genotoxic stress. Our results reveal a previously unknown role for nuclear actin filaments in DNA repair and identify the molecular mechanisms creating these nuclear filaments. In animals, plants, and other eukaryotic organisms, a cell's DNA is contained within a structure called the nucleus, which separates it from the rest of the interior of the cell. Filaments of a protein called actin are normally found outside the nucleus, where they help give the cell its overall shape and organize its contents. However, these filaments can sometimes form inside the nucleus in response to a sudden increase in heat or another type of stress. However, it was not clear what role these actin filaments play in the nucleus because it was difficult to distinguish them from the actin filaments that form in other parts of the cell. Researchers have recently developed new techniques to study actin filaments inside the nuclei of live cells under a microscope, using fluorescent protein tags. Here, Belin et al.—including some of the researchers involved in the previous work—used this technique to investigate whether DNA damage causes actin filaments to form in the nuclei of human cells. The experiments show that DNA damage does indeed lead to the formation of actin filaments in the nucleus. In a structure within the nucleus called the nucleolus, the actin filaments are short. However, in the rest of the nucleus, the actin forms long filaments and dense clusters. Cells that contained lower levels of actin were less able to repair their DNA than normal cells. Belin et al. also identified three proteins—called Formin-2, Spire-1, and Spire-2—that assemble the actin filaments in the nucleus. These proteins are also required to make actin filaments in other parts of the cell. The experiments show that the level of Formin-2 increases in the nucleus after DNA damage, and that the DNA of cells lacking this protein is more severely damaged. Belin et al.'s findings reveal a new role for actin in the repair of DNA and the next challenge is to understand the details of how this works.

Branched actin networks are self-assembling molecular motors that move biological membranes and drive many important cellular processes, including phagocytosis, endocytosis, and pseudopod protrusion. When confronted with opposing forces, the growth rate of these networks slows and their density increases, but the stoichiometry of key components does not change. The molecular mechanisms governing this force response are not well understood, so we used single-molecule imaging and AFM cantilever deflection to measure how applied forces affect each step in branched actin network assembly. Although load forces are observed to increase the density of growing filaments, we find that they actually decrease the rate of filament nucleation due to inhibitory interactions between actin filament ends and nucleation promoting factors. The force-induced increase in network density turns out to result from an exponential drop in the rate constant that governs filament capping. The force dependence of filament capping matches that of filament elongation and can be explained by expanding Brownian Ratchet theory to cover both processes. We tested a key prediction of this expanded theory by measuring the force-dependent activity of engineered capping protein variants and found that increasing the size of the capping protein increases its sensitivity to applied forces. In summary, we find that Brownian Ratchets underlie not only the ability of growing actin filaments to generate force but also the ability of branched actin networks to adapt their architecture to changing loads.

The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation requires the local control of membrane organization. Previous work has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin. We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors. Upon complete consumption of ATP, this system settles into distinct phases of actin organization, namely bundled filaments, linked apolar asters, and a lattice of polar asters. These depend on actin concentration, filament length, and actin/myosin ratio. During formation of the polar aster phase, advection of the self-organizing actomyosin network drives transient clustering of actin-associated membrane components. Regeneration of ATP supports a constitutively remodeling actomyosin state, which in turn drives active fluctuations of coupled membrane components, resembling those observed at the cell surface. In a multicomponent membrane bilayer, this remodeling actomyosin layer contributes to changes in the extent and dynamics of phase-segregating domains. These results show how local membrane composition can be driven by active processes arising from actomyosin, highlighting the fundamental basis of the active composite model of the cell surface, and indicate its relevance to the study of membrane organization.

The actin cytoskeleton is essential for many cellular functions including shape determination, intracellular transport and locomotion. Previous work has identified two factorsthe Arp2/3 complex and the formin family of proteinsthat nucleate new actin filaments via different mechanisms. Here we show that the Drosophila protein Spire represents a third class of actin nucleation factor. In vitro, Spire nucleates new filaments at a rate that is similar to that of the formin family of proteins but slower than in the activated Arp2/3 complex, and it remains associated with the slow-growing pointed end of the new filament. Spire contains a cluster of four WASP homology 2 (WH2) domains, each of which binds an actin monomer. Maximal nucleation activity requires all four WH2 domains along with an additional actin-binding motif, conserved among Spire proteins. Spire itself is conserved among metazoans and, together with the formin Cappuccino, is required for axis specification in oocytes and embryos, suggesting that multiple actin nucleation factors collaborate to construct essential cytoskeletal structures.

Type IV pili are filamentous appendages found in most bacteria and archaea, where they can support functions such as surface adhesion, DNA uptake, aggregation, and motility. In most bacteria, PilT-family ATPases disassemble adhesion pili, causing them to rapidly retract and produce twitching motility, important for surface colonization. As archaea do not possess PilT homologs, it was thought that archaeal pili cannot retract and that archaea do not exhibit twitching motility. Here, we use live-cell imaging, automated cell tracking, fluorescence imaging, and genetic manipulation to show that the hyperthermophilic archaeon Sulfolobus acidocaldarius exhibits twitching motility, driven by retractable adhesion (Aap) pili, under physiologically relevant conditions (75 °C, pH 2). Aap pili are thus capable of retraction in the absence of a PilT homolog, suggesting that the ancestral type IV pili in the last universal common ancestor (LUCA) were capable of retraction. Bacteria use filamentous appendages known as type IV pili for various functions, including twitching motility on surfaces. It was thought that archaeal pili cannot retract and that archaea do not exhibit twitching motility. However, Charles-Orszag et al. demonstrate that the model archaeon Sulfolobus acidocaldarius exhibits twitching motility, driven by retractable adhesion pili.

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (<0.75 µm), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that Arp2/3-dependent pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

Enabled/Vasodilator (Ena/VASP) proteins promote actin filament assembly at multiple locations, including: leading edge membranes, focal adhesions, and the surface of intracellular pathogens. One important Ena/VASP regulator is the mig-10/Lamellipodin/RIAM family of adaptors that promote lamellipod formation in fibroblasts and drive neurite outgrowth and axon guidance in neurons. To better understand how MRL proteins promote actin network formation we studied the interactions between Lamellipodin (Lpd), actin, and VASP, both in vivo and in vitro. We find that Lpd binds directly to actin filaments and that this interaction regulates its subcellular localization and enhances its effect on VASP polymerase activity. We propose that Lpd delivers Ena/VASP proteins to growing barbed ends and increases their polymerase activity by tethering them to filaments. This interaction represents one more pathway by which growing actin filaments produce positive feedback to control localization and activity of proteins that regulate their assembly. Actin—the most abundant protein in most eukaryotic cells—assembles into a network of filaments that spans the length and breadth of the cell. Like the skeleton of an animal, this ‘actin cytoskeleton’ gives the cell its shape and strength, and enables the cell to actively move through its environment. To start moving, many cells begin assembling actin filaments next to the cell membrane. The growth of these filaments pushes the membrane forward and creates a two-dimensional structure called a ‘lamellipod’, which explores the space around the cell and steers its movement. The actin filaments in a lamellipod are dynamic and undergo repeated cycles of assembly and disassembly. These processes are tightly regulated by a variety of other proteins. Members of the Ena/VASP protein family, for example, collect the building blocks of an actin filament and rapidly stack them in place on the fast-growing end of a filament. The activities of Ena/VASP proteins play an especially important role in creating lamellipodial actin networks and in driving cell movement. Previous work showed that a protein called Lamellipodin binds to Ena/VASP proteins and helps recruit them to the cell membrane. However, it was unclear whether Lamellipodin could affect the activity of Ena/VASP proteins or their interaction with the actin filaments. Hansen and Mullins have now analyzed the interactions between Ena/VASP, Lamellipodin and actin. The experiments demonstrate that Lamellipodin does not simply tether Ena/VASP proteins to the membrane but also binds directly to actin filaments, via a binding site that is distinct from the site that contacts Ena/VASP. Further experiments with purified proteins revealed that Lamellipodin could interact with both actin filaments and Ena/VASP proteins at the same time. Hansen and Mullins also found that purified Lamellipodin interacted with VASP proteins to form clustered protein complexes, and that together with the tethering of actin filaments to the membrane, this clustering greatly increased VASP's ability to lengthen actin filaments. By visualizing Lamellipodin tagged with a green fluorescent protein in living cells, Hansen and Mullins then showed that its interaction with actin filaments was sufficient to localize Lamellipodin to the cell membrane. Finally, since Lamellipodin interacts with a multitude of signaling molecules in addition to Ena/VASP proteins, the next big challenge is to understand how Lamellipodin itself is regulated. Future studies could also explore how cells harness the power of the actin cytoskeleton to carry out these essential activities.

The unfolded protein response (UPR) maintains protein folding homeostasis in the endoplasmic reticulum (ER). In metazoan cells, the Ire1 branch of the UPR initiates two functional outputs—non-conventional mRNA splicing and selective mRNA decay (RIDD). By contrast, Ire1 orthologs from Saccharomyces cerevisiae and Schizosaccharomyces pombe are specialized for only splicing or RIDD, respectively. Previously, we showed that the functional specialization lies in Ire1’s RNase activity, which is either stringently splice-site specific or promiscuous (Li et al., 2018). Here, we developed an assay that reports on Ire1’s RNase promiscuity. We found that conversion of two amino acids within the RNase domain of S. cerevisiae Ire1 to their S. pombe counterparts rendered it promiscuous. Using biochemical assays and computational modeling, we show that the mutations rewired a pair of salt bridges at Ire1 RNase domain’s dimer interface, changing its protomer alignment. Thus, Ire1 protomer alignment affects its substrates specificity.

Multiple unrelated polymer systems have evolved to partition DNA molecules between daughter cells at division. To better understand polymer-driven DNA segregation, we reconstituted the three-component segregation system of the R1 plasmid from purified components. We found that the ParR/parC complex can construct a simple bipolar spindle by binding the ends of ParM filaments, inhibiting dynamic instability, and acting as a ratchet permitting incorporation of new monomers and riding on the elongating filament ends. Under steady-state conditions, the dynamic instability of unattached ParM filaments provides the energy required to drive DNA segregation.