Comprehensive Analysis of Human Cytomegalovirus- and HIV-Mediated Plasma Membrane Remodeling in Macrophages.

The plasma membrane (PM) must be overcome by viruses during entry and release. Furthermore, the PM represents the cellular communication compartment and the immune system interface. Hence, viruses have evolved sophisticated strategies to remodel the PM, for instance to avoid immune sensing and clearance of infected cells. We performed a comprehensive analysis of cell surface dysregulation by two human-pathogenic viruses, human cytomegalovirus (HCMV) and human immunodeficiency virus type 1 (HIV-1), in primary macrophages, which are classical antigen-presenting cells and orchestrators of the immune system. Scanning ion conductance microscopy revealed a loss of roughness and an overall smooth phenotype of HCMV-infected macrophages, in contrast to HIV-1 infection. This phenotype was also evident on the molecular level. When we screened for cell surface receptors modulated by HCMV, 42 of 332 receptors tested were up- or downregulated, whereas HIV-1 affected only 7 receptors. In particular CD164, CD84, and CD180 were targeted by HCMV. Mechanistically, HCMV induced transcriptional silencing of these receptors in an interferon (IFN)-independent manner, and expression was reduced not only by lab-adapted HCMV but also by clinical HCMV isolates. Altogether, our plasma membrane profiling of human macrophages provides clues to understand how viruses evade the immune system and identified novel cell surface receptors targeted by HCMV. IMPORTANCE The PM is a key component that viruses have to cope with. It is a barrier for infection and egress and is critically involved in antiviral immune signaling. We hence asked the question how two immunomodulatory viruses, HIV-1 and HCMV, dysregulate this compartment in infected macrophages, relevant in vivo targets of both viruses. We employed a contact-free microscopic technique to image the PM of infected cells and performed a phenotypic flow cytometry-based screen to identify receptor modulations on a molecular level. Our results show that HIV-1 and HCMV differentially manipulate the PM of macrophages. While HIV-1-mediated changes are relatively subtle, HCMV induces major alterations of the PM. We identify novel immune receptors manipulated by HCMV and define mechanisms of how HCMV interferes with receptor expression. Altogether, our study reveals differential strategies of how two human-pathogenic viruses manipulate infected cells and identifies potential novel pathways of HCMV immune evasion.

Ultrafast Imaging of Cardiomyocyte Contractions by Combining Scanning Ion Conductance Microscopy with a Microelectrode Array.

Beating cardiomyocytes undergo fast morphodynamics during the contraction-relaxation cycle. However, imaging these morphodynamics with a high spatial and temporal resolution is difficult, owing to a lack of suitable techniques. Here, we combine scanning ion conductance microscopy (SICM) with a microelectrode array (MEA) to image the three-dimensional (3D) topography of cardiomyocytes during a contraction-relaxation cycle with 1 µm spatial and 1 ms time resolution. We record the vertical motion of cardiomyocytes at many locations across a cell by SICM and synchronize these data using the simultaneously recorded action potential by the MEA as a time reference. This allows us to reconstruct the time-resolved 3D morphology of cardiomyocytes during a full contraction-relaxation cycle with a raw data rate of 200 µs/frame and to generate spatially resolved images of contractile parameters (maximum displacement, time delay, asymmetry factor). We use the MEA-SICM setup to visualize the effect of blebbistatin, a myosin II inhibitor, on the morphodynamics of contractions. Further, we find an upper limit of 0.02% for cell volume changes during an action potential. The results show that MEA-SICM provides an ultrafast imaging platform for investigating the functional interplay of cardiomyocyte electrophysiology and mechanics.

High-speed scanning ion conductance microscopy for sub-second topography imaging of live cells.

Scanning ion conductance microscopy (SICM) is an emerging tool for non-invasive and high-resolution topography imaging of live cells. However, the imaging speed of conventional SICM setups is slow, requiring several seconds or even minutes per image, thereby making it difficult to study cellular dynamics. Here, we describe a high-speed SICM (HS-SICM) setup for topography imaging in the hopping mode with a pixel rate of 11.0 kHz, which is 15 times faster than what was reported before. In combination with a "turn step" procedure for rapid pipette retraction, we image the ultra-fast morphodynamics of live human platelets, A6 cells, and U2OS cells at a rate as fast as 0.6 s per frame. The results show that HS-SICM provides a useful platform for investigating the dynamics of cell morphology on a sub-second timescale.