Terrence M. Dobrowsky

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.

Single-Molecule Analysis of Human Immunodeficiency Virus Type 1 gp120-Receptor Interactions in Living Cells

ABSTRACT A quantitative description of the binding interactions between human immunodeficiency virus (HIV) type 1 envelope glycoproteins and their host cell surface receptors remains incomplete. Here, we introduce a single-molecule analysis that directly probes the binding interactions between an individual viral subunit gp120 and a single receptor CD4 and/or chemokine coreceptor CCR5 in living cells. This analysis differentiates single-molecule binding from multimolecule avidity and shows that, while the presence of CD4 is required for gp120 binding to CCR5, the force required to rupture a single gp120-coreceptor bond is significantly higher and its lifetime is much longer than those of a single gp120-receptor bond. The lifetimes of these bonds are themselves shorter than those of the P-selectin/PSGL-1 bond involved in leukocyte attachment to the endothelium bonds during an inflammation response. These results suggest an amended model of HIV entry in which, immediately after the association of gp120 to its receptor, gp120 seeks its coreceptor to rapidly form a new bond. This “bond transfer” occurs only if CCR5 is in close proximity to CD4 and CD4 is still attached to gp120. The analysis presented here may serve as a general framework to study mechanisms of receptor-mediated interactions between viral envelope proteins and host cell receptors at the single-molecule level in living cells.

Monitoring Early Fusion Dynamics of Human Immunodeficiency Virus Type 1 at Single-Molecule Resolution

ABSTRACT The fusion of human immunodeficiency virus type 1 (HIV-1) to host cells is a dynamic process governed by the interaction between glycoproteins on the viral envelope and the major receptor, CD4, and coreceptor on the surface of the cell. How these receptors organize at the virion-cell interface to promote a fusion-competent site is not well understood. Using single-molecule force spectroscopy, we map the tensile strengths, lifetimes, and energy barriers of individual intermolecular bonds between CCR5-tropic HIV-1 gp120 and its receptors CD4 and CCR5 or CXCR4 as a function of the interaction time with the cell. According to the Bell model, at short times of contact between cell and virion, the gp120-CD4 bond is able to withstand forces up to 35 pN and has an initial lifetime of 0.27 s and an intermolecular length of interaction of 0.34 nm. The initial bond also has an energy barrier of 6.7 kBT (where kB is Boltzmanns constant and T is absolute temperature). However, within 0.3 s, individual gp120-CD4 bonds undergo rapid destabilization accompanied by a shortened lifetime and a lowered tensile strength. This destabilization is significantly enhanced by the coreceptor CCR5, not by CXCR4 or fusion inhibitors, which suggests that it is directly related to a conformational change in the gp120-CD4 bond. These measurements highlight the instability and low tensile strength of gp120-receptor bonds, uncover a synergistic role for CCR5 in the progression of the gp120-CD4 bond, and suggest that the cell-virus adhesion complex is functionally arranged about a long-lived gp120-coreceptor bond.

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.

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.

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.

Chapter 15: Live-cell single-molecule force spectroscopy.

We describe a method to measure the kinetics and micromechanical properties of individual receptor-ligand bonds formed between two living cells. Using living cells rather than recombinant proteins ensures that the orientation, surface density, and posttranslational modifications of the probed receptors are physiological and that their regulated attachment to the cytoskeleton can occur. A cell is tethered to a flexible cantilever and brought into contact with cells adherent to a substratum before being pulled at a controlled retraction velocity. Measurements of bond rupture forces and associated bond loading rates over an extended range of retraction velocities allow us to compute precisely the tensile strength, reactive compliance, lifetime, and dissociation rate of individual intercellular receptor-ligand bonds. We also describe tests of specificity and Monte Carlo simulations, which ensure that measurements obtained by this method correspond to a single type of intercellular adhesion bond. We illustrate this live-cell single molecule force spectroscopy assay by characterizing homotypic bonds composed of vascular endothelial -cadherin pairs formed between living endothelial cells. This versatile assay could be used to establish the molecular principles that drive a wide range of important physiological processes involving receptor-mediated intercellular adhesion, such as the immunological synapse between a lymphocyte and an antigen-presenting cell and synaptic interactions between neuron cells, and pathological processes resulting in altered intercellular adhesion.

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.

Single-Molecule Analysis of Cell-Virus Binding Interactions

Adhesion assays based on single molecule interactions are a useful option when discerning between avidity and affinity in complex systems. This is especially true for viral adhesion to living cells which typically involves a complex system of proteins working together to lead to productive infection. Here, we discuss assays that have been used to quantitatively study the adhesion of viral and cellular receptors including surface plasmon resonance, real time fusion assays involving viral fluorescent tags and single molecule force spectroscopy (SMFS). We highlight the advantages of SMFS over other methods, including its specificity, versatility and application to studying the adhesion of HIV-1 to human cells. We discuss how using SMFS with infectious virus and living cells allow us to distinguish the adhesion of HIV-1 surface protein, gp120, to its primary cellular receptor, CD4, from the adhesion of gp120 to its secondary co-receptor, CCR5.