High-resolution mass measurements of single budding yeast reveal linear growth segments.

The regulation of cell growth has fundamental physiological, biotechnological and medical implications. However, methods that can continuously monitor individual cells at sufficient mass and time resolution hardly exist. Particularly, detecting the mass of individual microbial cells, which are much smaller than mammalian cells, remains challenging. Here, we modify a previously described cell balance ('picobalance') to monitor the proliferation of single cells of the budding yeast, Saccharomyces cerevisiae, under culture conditions in real time. Combined with optical microscopy to monitor the yeast morphology and cell cycle phase, the picobalance approaches a total mass resolution of 0.45 pg. Our results show that single budding yeast cells (S/G2/M phase) increase total mass in multiple linear segments sequentially, switching their growth rates. The growth rates weakly correlate with the cell mass of the growth segments, and the duration of each growth segment correlates negatively with cell mass. We envision that our technology will be useful for direct, accurate monitoring of the growth of single cells throughout their cycle.

Detecting molecular interactions that stabilize native bovine rhodopsin.

Using single-molecule force spectroscopy we probed molecular interactions within native bovine rhodopsin and discovered structural segments of well-defined mechanical stability. Highly conserved residues among G protein-coupled receptors were located at the interior of individual structural segments, suggesting a dual role for these segments in rhodopsin. Firstly, structural segments stabilize secondary structure elements of the native protein, and secondly, they position and hold the highly conserved residues at functionally important environments. Two main classes of force curves were observed. One class corresponded to the unfolding of rhodopsin with the highly conserved Cys110-Cys187 disulfide bond remaining intact and the other class corresponded to the unfolding of the entire rhodopsin polypeptide chain. In the absence of the Cys110-Cys187 bond, the nature of certain molecular interactions within folded rhodopsin was altered. These changes highlight the structural importance of this disulfide bond and may form the basis of dysfunctions associated with its absence.

Roll, adhere, spread and contract: structural mechanics of platelet function.

Platelets are involved in life-sustaining processes such as hemostasis, wound healing, atherothrombosis and angiogenesis. Mechanical trauma to blood vessels causes platelet activation resulting in their adherence and clot formation at the damaged site, culminating in clot retraction and tissue repair. Two of the major players underlying this process are the cytoskeleton, i.e., actin and microtubules, and the membrane integrin receptors. Rare congenital bleeding disorders such as Glanzmann thrombasthenia and Bernard-Soulier syndrome are associated with genetic alterations of platelet surface receptors, also affecting the platelet cytoskeletal structure. In this review, we summarize the current knowledge about platelet structure and adhesion, and delve into the mechanical aspects of platelet function. Platelets lack a nucleus, and can thus provide a minimal model of a biological cell. New biophysical tools may help to scrutinize platelets anew and to extend the existing knowledge on cell biology.

A droplet microfluidic system for sequential generation of lipid bilayers and transmembrane electrical recordings.

This paper demonstrates a microfluidic system that automates i) formation of a lipid bilayer at the interface between a pair of nanoliter-sized aqueous droplets in oil, ii) exchange of one droplet of the pair to form a new bilayer, and iii) current measurements on single proteins. A new microfluidic architecture is introduced - a set of traps designed to localize the droplets with respect to each other and with respect to the recording electrodes. The system allows for automated execution of experimental protocols by active control of the flow on chip with the use of simple external valves. Formation of stable artificial lipid bilayers, incorporation of α-hemolysin into the bilayers and electrical measurements of ionic transport through the protein pore are demonstrated.