Initial Step of Virus Entry: Virion Binding to Cell-Surface Glycans

Virus infection is an intricate process that requires the concerted action of both viral and host cell components. Entry of viruses into cells is initiated by interactions between viral proteins and cell-surface receptors. Various cell-surface glycans function as initial, usually low-affinity attachment factors, providing a first anchor of the virus to the cell surface, and further facilitate high-affinity binding to virus-specific cell-surface receptors, while other glycans function as specific entry receptors themselves. It is now possible to rapidly identify specific glycan receptors using different techniques, define atomic-level structures of virus-glycan complexes, and study these interactions at the single-virion level. This review provides a detailed overview of the role of glycans in viral infection and highlights experimental approaches to study virus-glycan binding along with specific examples. In particular, we highlight the development of the atomic force microscope to investigate interactions with glycans at the single-virion level directly on living mammalian cells, which offers new perspectives to better understand virus-glycan interactions in physiologically relevant conditions.

A Role for Septins in the Interaction between the Listeria monocytogenes Invasion Protein InlB and the Met Receptor

Sandor Kasas, Charles Roduit

Septins are conserved GTPases that form filaments and are required for cell division. During interphase, septin filaments associate with cellular membrane and cytoskeleton networks, yet the functional significance of these associations have, to our knowledge, remained unknown. We recently discovered that different septins, SEPT2 and SEPT11, regulate the InIB-mediated entry of Listeria monocytogenes into host cells. Here we address the role of SEPT2 and SEPT11 in the InIB-Met interactions underlying Listeria invasion to explore how septins modulate surface receptor function. We observed that differences in InIB-mediated Listeria entry correlated with differences in Met surface expression caused by septin depletion. Using atomic force microscopy on living cells, we show that septin depletion significantly reduced the unbinding force of InIB-Met interaction and the viscosity of membrane tethers at locations where the InIB-Met interaction occurs. Strikingly, the same order of difference was observed for cells in which the actin cytoskeleton was disrupted. Consistent with a proposed role of septins in association with the actin cytoskeleton, we show that cell elasticity is decreased upon septin or actin inactivation. Septins are therefore likely to participate in anchorage of the Met receptor to the actin cytoskeleton, and represent a critical determinant in surface receptor function.

Elsevier2011

Structural basis and biophysical properties of synaptic plasticity studied by atomic force microscopy

Alexandre Yersin

In this thesis, we studied two systems important for synaptic plasticity, one presynaptic and another postsynaptic. The protein complex composed of VAMP 2, SNAP-25 and syntaxin 1 (SNARE complex) is essential for docking and fusion of neurotransmitter-filled synaptic vesicles with the presynaptic membrane. In order to better understand the fusion mechanisms, we reconstituted the synaptic SNARE complex in the imaging chamber of an atomic force microscope (AFM) and measured the interaction forces between its components. Each protein was tested against the two others, taken either individually or as binary complexes. This approach allowed us to determine specific interaction forces, dissociation kinetics of the SNAREs and led us to propose a sequence of formation for the complex. A theoretical model based on our measurements suggests that a minimum of four complexes is probably necessary for fusion to occur. We also showed that the regulatory protein nSec1 injected into the AFM chamber prevented the SNARE complex formation. Finally we measured the effect of tetanus toxin protease (TeTx) on the SNARE complex and its activity by on-line registration during TeTx injection. These experiments reveal that AFM is a valuable tool to investigate complicate interactions among a protein complex containing up to four components. Trafficking of glutamate receptors AMPA is closely related to postsynaptic induction of long-term potentiation. In a second part of this thesis, we used AFM to explore the distribution of AMPA receptors on-line at the surface of living cells in correlation with cellular viscoelastic properties. First, we showed that AFM permits to detect specifically AMPA receptors at the surface of living neurons and we proved that the specificity of detection is stable during time. Moreover, we were able to visualize variations in AMPA receptor density on surface in response to different stimulations. Finally, AFM also allowed us to study local viscoelastic properties of the cell and their modifications occurring during AMPA receptor trafficking. Our measurements reveal that AMPA receptors are situated at the cell surface on local stiffness maxima. We have noticed that an excitatory stimulation removes these stiffness maxima before producing internalization of receptors. Our results suggest also that AMPA receptors may be constituted of two subpopulations depending on the cell stiffness. A "hard" subpopulation may be preferentially internalized after an excitatory stimulation whereas a "soft" one seems to remain at the cell surface.

EPFL2004

Cellular dynamics explored with digital holographic microscopy

Benjamin Rappaz

Obtaining high resolution 3D quantitative images is critical for elucidating the relationship between cell dynamics and physiological or pathological processes. However the feasibility of such cellular dynamic studies is limited by the resolution of the 3D images, the required acquisition time and the degree of invasiveness of the currently available imaging techniques as well as their limitation to provide quantitative information. Digital Holographic Microscopy (DHM) is an optical imaging technique developed to meet most of these requirements to accurately explore cell dynamics. In short, DHM allows to quantitatively measure the wavefront transmitted through a specimen and magnified by a microscope objective. A hologram, which contains the whole information of the transmitted wavefront, is formed by the interference of the wave coming from the object with a reference wave, and recorded with a camera. The hologram reconstruction process, based on the electromagnetic wave propagation theory, permits to numerically process the hologram in order to extract both amplitude and phase information. This process allows to obtain an exact replica of the wave diffracted or transmitted by the observed specimen. Thanks to its interferometric nature, DHM yields phase images with a nanometric sensitivity along the optical axis of the microscope, revealing extremely detailed minute information about the cell morphology and internal structure, as well as cellular micromovements in the nanometer range. So far, DHM has proven its efficiency on numerous fields in material sciences. This thesis presents some technical developments achieved with DHM and extends the applications of this technique to life sciences in general and cellular structure and dynamics in particular. The quantitative phase images generated by DHM represent an endogenous contrast signal which contains dual information about both the cell morphology and the refractive index (RI), which is essentially related to the intracellular (protein) content. The first part of this thesis (chapter 2) presents two different approaches to separately extract these two contributions and obtain an independent measurement of both the cell morphology and RI, thus providing an interpretation of the phase signal which is more readily exploitable and more relevant for cell biologists. The RI is a function of the dry mass, thus the phase signal variation in time can be related to biomass production and provides a direct interpretation of the phase signal. In section 3.1 this relation is exploited to study the dry mass production during the cell cycle of fission yeast. The decoupling procedure presented in section 2.1 allows to measure the thickness and RI of cells. In section 3.2, we use this decoupling procedure to measure the Mean Corpuscular Volume (MCV) and RI of red blood cells. These measurements are also compared to those obtained by other techniques. In addition, as far as red blood cell are concerned, the RI can be related to the Mean Corpuscular Hemoglobin Content (MCHC). The MCV, RI and MCHC are important clinical parameters that can be non-invasively and instantly measured with DHM. The membrane of red cells presents a flickering at a nanometric scale which can reflect their metabolic state, related to ATP content. Due to the technical difficulty to measure this flickering, it has been difficult to investigate the origin of these fluctuations. DHM, thanks to its nanometric and microsecond sensitivity, allows to monitor these Cell Membrane Fluctuations (CMF). In section 3.3 DHM is used to measure CMF of healthy and metabolically-impaired red blood cells. The last section of this thesis (section 3.4) assesses how DHM can quantitatively monitor minute cell morphological and optical changes induced by agonist-mediated activation of specific membrane receptors. This is illustrated by tracking the phase signal variations during excitotoxic activation of neurons by the neurotransmitter glutamate.