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Abstract Many bacteria inhabit thin layers of water on solid surfaces both naturally in soils or on hosts or textiles and in the lab on agar hydrogels. In these environments, cells experience capillary forces, yet an understanding of how these forces shape bacterial collective behaviors remains elusive. Here, we show that the water menisci formed around bacteria lead to capillary attraction between cells while still allowing them to slide past one another. We develop an experimental apparatus that allows us to control bacterial collective behaviors by varying the strength and range of capillary forces. Combining 3D imaging and cell tracking with agent-based modeling, we demonstrate that capillary attraction organizes rod-shaped bacteria into densely packed, nematic groups, and profoundly influences their collective dynamics and morphologies. Our results suggest that capillary forces may be a ubiquitous physical ingredient in shaping microbial communities in partially hydrated environments. 

ABSTRACT The crowded bacterial cytoplasm is comprised of biomolecules that span several orders of magnitude in size and electrical charge. This complexity has been proposed as the source of the rich spatial organization and apparent anomalous diffusion of intracellular components, although this has not been tested directly. Here, we use biplane microscopy to track the 3D motion of self-assembled bacterial Genetically Encoded Multimeric nanoparticles (bGEMs) with tunable size (20 to 50 nm) and charge (−2160 to +1800 e) in liveEscherichia colicells. To probe intermolecular details at spatial and temporal resolutions beyond experimental limits, we also developed a colloidal whole-cell model that explicitly represents the size and charge of cytoplasmic macromolecules and the porous structure of the bacterial nucleoid. Combining these techniques, we show that bGEMs spatially segregate by size, with small 20-nm particles enriched inside the nucleoid, and larger and/or positively charged particles excluded from this region. Localization is driven by entropic and electrostatic forces arising from cytoplasmic polydispersity, nucleoid structure, geometrical confinement, and interactions with other biomolecules including ribosomes and DNA. We observe that at the timescales of traditional single molecule tracking experiments, motion appears sub-diffusive for all particle sizes and charges. However, using computer simulations with higher temporal resolution, we find that the apparent anomalous exponents are governed by the region of the cell in which bGEMs are located. Molecular motion does not display anomalous diffusion on short time scales and the apparent sub-diffusion arises from geometrical confinement within the nucleoid and by the cell boundary. 

Abstract Microtubules are generated at centrosomes, chromosomes, and within spindles during cell division. Whereas microtubule nucleation at the centrosome is well characterized, much remains unknown about where, when, and how microtubules are nucleated at chromosomes. To address these questions, we reconstitute microtubule nucleation from purified chromosomes in meiotic Xenopus egg extract and find that chromosomes alone can form spindles. We visualize microtubule nucleation near chromosomes using total internal reflection fluorescence microscopy to find that this occurs through branching microtubule nucleation. By inhibiting molecular motors, we find that the organization of the resultant polar branched networks is consistent with a theoretical model where the effectors for branching nucleation are released by chromosomes, forming a concentration gradient that spatially biases branching microtbule nucleation. In the presence of motors, these branched networks are ultimately organized into functional spindles, where the number of emergent spindle poles scales with the number of chromosomes and total chromatin area. 

Abstract Multifocal microscopy enables high-speed three-dimensional (3D) volume imaging by using a multifocal grating in the emission path. This grating is typically designed to afford a uniform illumination of multifocal subimages for a single emission wavelength. Using the same grating for multicolor imaging results in non-uniform subimage intensities in emission wavelengths for which the grating is not designed. This has restricted multifocal microscopy applications for samples having multicolored fluorophores. In this paper, we present a multicolor multifocal microscope implementation which uses a Spatial Light Modulator (SLM) as a single multifocal grating to realize near-uniform multifocal subimage intensities across multiple wavelength emission bands. Using real-time control of an in-situ-optimized SLM implemented as a multifocal grating, we demonstrate multicolor multifocal 3D imaging over three emission bands by imaging multicolored particles as well as Escherichia coli ( E. coli ) interacting with human liver cancer cells, at $$\sim 2.5$$ ∼ 2.5 multicolor 3D volumes per second acquisition speed. Our multicolor multifocal method is adaptable across SLM hardware, emission wavelength band locations and number of emission bands, making it particularly suited for researchers investigating fast processes occurring across a volume where multiple species are involved. 

Abstract The cerebellum regulates nonmotor behavior, but the routes of influence are not well characterized. Here we report a necessary role for the posterior cerebellum in guiding a reversal learning task through a network of diencephalic and neocortical structures, and in flexibility of free behavior. After chemogenetic inhibition of lobule VI vermis or hemispheric crus I Purkinje cells, mice could learn a water Y-maze but were impaired in ability to reverse their initial choice. To map targets of perturbation, we imaged c-Fos activation in cleared whole brains using light-sheet microscopy. Reversal learning activated diencephalic and associative neocortical regions. Distinctive subsets of structures were altered by perturbation of lobule VI (including thalamus and habenula) and crus I (including hypothalamus and prelimbic/orbital cortex), and both perturbations influenced anterior cingulate and infralimbic cortex. To identify functional networks, we used correlated variation in c-Fos activation within each group. Lobule VI inactivation weakened within-thalamus correlations, while crus I inactivation divided neocortical activity into sensorimotor and associative subnetworks. In both groups, high-throughput automated analysis of whole-body movement revealed deficiencies in across-day behavioral habituation to an open-field environment. Taken together, these experiments reveal brainwide systems for cerebellar influence that affect multiple flexible responses. 

Abstract In mammals, subcellular protein localization of factors like planar cell polarity proteins is a key driver of the multicellular organization of tissues. Bacteria also form organized multicellular communities, but these patterns are largely thought to emerge from regulation of whole-cell processes like growth, motility, cell shape, and differentiation. Here we show that a unique intracellular patterning of appendages known as type IV pili (T4P) can drive multicellular development of complex bacterial communities. Specifically, dynamic T4P appendages localize in a line along the long axis of the cell in the bacterium Acinetobacter baylyi . This long-axis localization is regulated by a functionally divergent chemosensory Pil-Chp system, and an atypical T4P protein homologue (FimV) bridges Pil-Chp signaling and T4P positioning. We further demonstrate through modeling and empirical approaches that subcellular T4P localization controls how individual cells interact with one another, independently of T4P dynamics, with different patterns of localization giving rise to distinct multicellular architectures. Our results reveal how subcellular patterning of single cells regulates the development of multicellular bacterial communities. 

The ability of eukaryotic cells to differentiate surface stiffness is fundamental for many processes like stem cell development. Bacteria were previously known to sense the presence of surfaces, but the extent to which they could differentiate stiffnesses remained unclear. Here we establish that the human pathogen Pseudomonas aeruginosa actively measures surface stiffness using type IV pili (TFP). Stiffness sensing is nonlinear, as induction of the virulence factor regulator is peaked with stiffness in a physiologically important range between 0.1 kPa (similar to mucus) and 1,000 kPa (similar to cartilage). Experiments on surfaces with distinct material properties establish that stiffness is the specific biophysical parameter important for this sensing. Traction force measurements reveal that the retraction of TFP is capable of deforming even stiff substrates. We show how slow diffusion of the pilin PilA in the inner membrane yields local concentration changes at the base of TFP during extension and retraction that change with substrate stiffness. We develop a quantitative biomechanical model that explains the transcriptional response to stiffness. A competition between PilA diffusion in the inner membrane and a loss/gain of monomers during TFP extension/retraction produces substrate stiffness-dependent dynamics of the local PilA concentration. We validated this model by manipulating the ATPase activity of the TFP motors to change TFP extension and retraction velocities and PilA concentration dynamics, altering the stiffness response in a predictable manner. Our results highlight stiffness sensing as a shared behavior across biological kingdoms, revealing generalizable principles of environmental sensing across small and large cells.