Brian R. Daniels

Condensation of FtsZ filaments can drive bacterial cell division

Forces are important in biological systems for accomplishing key cell functions, such as motility, organelle transport, and cell division. Currently, known force generation mechanisms typically involve motor proteins. In bacterial cells, no known motor proteins are involved in cell division. Instead, a division ring (Z-ring) consists of mostly FtsZ, FtsA, and ZipA is used to exerting a contractile force. The mechanism of force generation in bacterial cell division is unknown. Using computational modeling, we show that Z-ring formation results from the colocalization of FtsZ and FtsA mediated by the favorable alignment of FtsZ polymers. The model predicts that the Z-ring undergoes a condensation transition from a low-density state to a high-density state and generates a sufficient contractile force to achieve division. FtsZ GTP hydrolysis facilitates monomer turnover during the condensation transition, but does not directly generate forces. In vivo fluorescence measurements show that FtsZ density increases during division, in accord with model results. The mechanism is akin to van der Waals picture of gas-liquid condensation, and shows that organisms can exploit microphase transitions to generate mechanical forces.

Plasticity of the asialoglycoprotein receptor deciphered by ensemble FRET imaging and single-molecule counting PALM imaging

The stoichiometry and composition of membrane protein receptors are critical to their function. However, the inability to assess receptor subunit stoichiometry in situ has hampered efforts to relate receptor structures to functional states. Here, we address this problem for the asialoglycoprotein receptor using ensemble FRET imaging, analytical modeling, and single-molecule counting with photoactivated localization microscopy (PALM). We show that the two subunits of asialoglycoprotein receptor [rat hepatic lectin 1 (RHL1) and RHL2] can assemble into both homo- and hetero-oligomeric complexes, displaying three forms with distinct ligand specificities that coexist on the plasma membrane: higher-order homo-oligomers of RHL1, higher-order hetero-oligomers of RHL1 and RHL2 with two-to-one stoichiometry, and the homo-dimer RHL2 with little tendency to further homo-oligomerize. Levels of these complexes can be modulated in the plasma membrane by exogenous ligands. Thus, even a simple two-subunit receptor can exhibit remarkable plasticity in structure, and consequently function, underscoring the importance of deciphering oligomerization in single cells at the single-molecule level.

Asymmetric enrichment of PIE-1 in the Caenorhabditis elegans zygote mediated by binary counterdiffusion

To generate cellular diversity in developing organisms while simultaneously maintaining the developmental potential of the germline, germ cells must be able to preferentially endow germline daughter cells with a cytoplasmic portion containing specialized cell fate determinants not inherited by somatic cells. In Caenorhabditis elegans, germline inheritance of the protein PIE-1 is accomplished by first asymmetrically localizing the protein to the germplasm before cleavage and subsequently degrading residual levels of the protein in the somatic cytoplasm after cleavage. Despite its critical involvement in cell fate determination, the enrichment of germline determinants remains poorly understood. Here, combining live-cell fluorescence methods and kinetic modeling, we demonstrate that the enrichment process does not involve protein immobilization, intracellular compartmentalization, or localized protein degradation. Instead, our results support a heterogeneous reaction/diffusion model for PIE-1 enrichment in which the diffusion coefficient of PIE-1 is reversibly reduced in the posterior, resulting in a stable protein gradient across the zygote at steady state.

Probing Cellular Mechanical Responses to Stimuli Using Ballistic Intracellular Nanorheology

We describe a new method to measure the local and global micromechanical properties of the cytoplasm of single living cells in their physiological milieu and subjected to external stimuli. By tracking spontaneous, Brownian movements of individual nanoparticles of diameter>or=100 nm distributed within the cell with high spatial and temporal resolutions, the local viscoelastic properties of the intracellular milieu can be measured in different locations within the cell. The amplitude and the time-dependence of the mean-squared displacement of each nanoparticle directly reflect the elasticity and the viscosity of the cytoplasm in the vicinity of the nanoparticle. In our previous versions of particle tracking, we delivered nanoparticles via microinjection, which limited the number of cells amenable to measurement, rendering our technique incompatible with high-throughput experiments. Here we introduce ballistic injection to effectively deliver a large number of nanoparticles to a large number of cells simultaneously. When coupled with multiple particle tracking, this new method-ballistic intracellular nanorheology (BIN)-makes it now possible to probe the viscoelastic properties of cells in high-throughput experiments, which require large quantities of injected cells for seeding in various conditions. For instance, BIN allows us to probe an ensemble of cells embedded deeply inside a three-dimensional extracellular matrix or as a monolayer of cells subjected to shear flows.

MEX-5 enrichment in the C. elegans early embryo mediated by differential diffusion

Specification of germline and somatic cell lineages in C. elegans originates in the polarized single-cell zygote. Several cell-fate determinants are partitioned unequally along the anterior-posterior axis of the zygote, ensuring the daughter cells a unique inheritance upon asymmetric cell division. Recent studies have revealed that partitioning of the germline determinant PIE-1 and the somatic determinant MEX-5 involve protein redistribution accompanied by spatiotemporal changes in protein diffusion rates. Here, we characterize the dynamics of MEX-5 in the zygote and propose a novel reaction/diffusion model to explain both its anterior enrichment and its remarkable intracellular dynamics without requiring asymmetrically distributed binding sites. We propose that asymmetric cortically localized PAR proteins mediate the anterior enrichment of MEX-5 by reversibly changing its diffusion rate at spatially distinct points in the embryo, thus generating a stable concentration gradient along the anterior-posterior axis of the cell. This work extends the scope of reaction/diffusion models to include not only germline morphogens, but also somatic determinants.

SMRT analysis of MTOC and nuclear positioning reveals the role of EB1 and LIC1 in single-cell polarization

In several migratory cells, the microtubule-organizing center (MTOC) is repositioned between the leading edge and nucleus, creating a polarized morphology. Although our understanding of polarization has progressed as a result of various scratch-wound and cell migration studies, variations in culture conditions required for such assays have prevented a unified understanding of the intricacies of MTOC and nucleus positioning that result in cell polarization. Here, we employ a new SMRT (for sparse, monolayer, round, triangular) analysis that uses a universal coordinate system based on cell centroid to examine the pathways regulating MTOC and nuclear positions in cells plated in a variety of conditions. We find that MTOC and nucleus positioning are crucially and independently affected by cell shape and confluence; MTOC off-centering correlates with the polarization of single cells; acto-myosin contractility and microtubule dynamics are required for single-cell polarization; and end binding protein 1 and light intermediate chain 1, but not Par3 and light intermediate chain 2, are required for single-cell polarization and directional cell motility. Using various cellular geometries and conditions, we implement a systematic and reproducible approach to identify regulators of MTOC and nucleus positioning that depend on extracellular guidance cues.

Organization of Cellular Receptors into a Nanoscale Junction during HIV-1 Adhesion

The fusion of the human immunodeficiency virus type 1 (HIV-1) with its host cell is the target for new antiretroviral therapies. Viral particles interact with the flexible plasma membrane via viral surface protein gp120 which binds its primary cellular receptor CD4 and subsequently the coreceptor CCR5. However, whether and how these receptors become organized at the adhesive junction between cell and virion are unknown. Here, stochastic modeling predicts that, regarding binding to gp120, cellular receptors CD4 and CCR5 form an organized, ring-like, nanoscale structure beneath the virion, which locally deforms the plasma membrane. This organized adhesive junction between cell and virion, which we name the viral junction, is reminiscent of the well-characterized immunological synapse, albeit at much smaller length scales. The formation of an organized viral junction under multiple physiopathologically relevant conditions may represent a novel intermediate step in productive infection.

Multiscale diffusion in the mitotic Drosophila melanogaster syncytial blastoderm

Despite the fundamental importance of diffusion for embryonic morphogen gradient formation in the early Drosophila melanogaster embryo, there remains controversy regarding both the extent and the rate of diffusion of well-characterized morphogens. Furthermore, the recent observation of diffusional “compartmentalization” has suggested that diffusion may in fact be nonideal and mediated by an as-yet-unidentified mechanism. Here, we characterize the effects of the geometry of the early syncytial Drosophila embryo on the effective diffusivity of cytoplasmic proteins. Our results demonstrate that the presence of transient mitotic membrane furrows results in a multiscale diffusion effect that has a significant impact on effective diffusion rates across the embryo. Using a combination of live-cell experiments and computational modeling, we characterize these effects and relate effective bulk diffusion rates to instantaneous diffusion coefficients throughout the syncytial blastoderm nuclear cycle phase of the early embryo. This multiscale effect may be related to the effect of interphase nuclei on effective diffusion, and thus we propose that an as-yet-unidentified role of syncytial membrane furrows is to temporally regulate bulk embryonic diffusion rates to balance the multiscale effect of interphase nuclei, which ultimately stabilizes the shapes of various morphogen gradients.

Adhesion and fusion efficiencies of human immunodeficiency virus type 1 (HIV-1) surface proteins

In about half of patients infected with HIV-1 subtype B, viral populations shift from utilizing the transmembrane protein CCR5 to CXCR4, as well as or instead of CCR5, during late stage progression of the disease. How the relative adhesion efficiency and fusion competency of the viral Env proteins relate to infection during this transition is not well understood. Using a virus-cell fusion assay and live-cell single-molecule force spectroscopy, we compare the entry competency of viral clones to tensile strengths of the individual Env-receptor bonds of Env proteins obtained from a HIV-1 infected patient prior to and during coreceptor switching. The results suggest that the genetic determinants of viral entry were predominantly enriched in the C3, HR1 and CD regions rather than V3. Env proteins can better mediate entry into cells after coreceptor switch; this effective entry capacity does not correlate with the bond strengths between viral Env and cellular receptors.