Lateral plasma membrane compartmentalization links protein function and turnover.

Biological membranes organize their proteins and lipids into nano- and microscale patterns. In the yeast plasma membrane (PM), constituents segregate into a large number of distinct domains. However, whether and how this intricate patchwork contributes to biological functions at the PM is still poorly understood. Here, we reveal an elaborate interplay between PM compartmentalization, physiological function, and endocytic turnover. Using the methionine permease Mup1 as model system, we demonstrate that this transporter segregates into PM clusters. Clustering requires sphingolipids, the tetraspanner protein Nce102, and signaling through TORC2. Importantly, we show that during substrate transport, a simple conformational change in Mup1 mediates rapid relocation into a unique disperse network at the PM Clustered Mup1 is protected from turnover, whereas relocated Mup1 actively recruits the endocytic machinery thereby initiating its own turnover. Our findings suggest that lateral compartmentalization provides an important regulatory link between function and turnover of PM proteins.

Cytokinesis in vertebrate cells initiates by contraction of an equatorial actomyosin network composed of randomly oriented filaments.

The actomyosin ring generates force to ingress the cytokinetic cleavage furrow in animal cells, yet its filament organization and the mechanism of contractility is not well understood. We quantified actin filament order in human cells using fluorescence polarization microscopy and found that cleavage furrow ingression initiates by contraction of an equatorial actin network with randomly oriented filaments. The network subsequently gradually reoriented actin filaments along the cell equator. This strictly depended on myosin II activity, suggesting local network reorganization by mechanical forces. Cortical laser microsurgery revealed that during cytokinesis progression, mechanical tension increased substantially along the direction of the cell equator, while the network contracted laterally along the pole-to-pole axis without a detectable increase in tension. Our data suggest that an asymmetric increase in cortical tension promotes filament reorientation along the cytokinetic cleavage furrow, which might have implications for diverse other biological processes involving actomyosin rings.

The nuclear F-actin interactome of Xenopus oocytes reveals an actin-bundling kinesin that is essential for meiotic cytokinesis.

Nuclei of Xenopus laevis oocytes grow 100 000-fold larger in volume than a typical somatic nucleus and require an unusual intranuclear F-actin scaffold for mechanical stability. We now developed a method for mapping F-actin interactomes and identified a comprehensive set of F-actin binders from the oocyte nuclei. Unexpectedly, the most prominent interactor was a novel kinesin termed NabKin (Nuclear and meiotic actin-bundling Kinesin). NabKin not only binds microtubules but also F-actin structures, such as the intranuclear actin bundles in prophase and the contractile actomyosin ring during cytokinesis. The interaction between NabKin and F-actin is negatively regulated by Importin-ß and is responsive to spatial information provided by RanGTP. Disconnecting NabKin from F-actin during meiosis caused cytokinesis failure and egg polyploidy. We also found actin-bundling activity in Nabkin's somatic paralogue KIF14, which was previously shown to be essential for somatic cell division. Our data are consistent with the notion that NabKin/KIF14 directly link microtubules with F-actin and that such link is essential for cytokinesis.

From mosaic to patchwork: matching lipids and proteins in membrane organization.

Biological membranes encompass and compartmentalize cells and organelles and are a prerequisite to life as we know it. One defining feature of membranes is an astonishing diversity of building blocks. The mechanisms and principles organizing the thousands of proteins and lipids that make up membrane bilayers in cells are still under debate. Many terms and mechanisms have been introduced over the years to account for certain phenomena and aspects of membrane organization and function. Recently, the different viewpoints - focusing on lipids vs. proteins or physical vs. molecular driving forces for membrane organization - are increasingly converging. Here we review the basic properties of biological membranes and the most common theories for lateral segregation of membrane components before discussing an emerging model of a self-organized, multi-domain membrane or 'patchwork membrane'.

Visualization of cortex organization and dynamics in microorganisms, using total internal reflection fluorescence microscopy.

TIRF microscopy has emerged as a powerful imaging technology to study spatio-temporal dynamics of fluorescent molecules in vitro and in living cells. The optical phenomenon of total internal reflection occurs when light passes from a medium with high refractive index into a medium with low refractive index at an angle larger than a characteristic critical angle (i.e. closer to being parallel with the boundary). Although all light is reflected back under such conditions, an evanescent wave is created that propagates across and along the boundary, which decays exponentially with distance, and only penetrates sample areas that are 100-200 nm near the interface. In addition to providing superior axial resolution, the reduced excitation of out of focus fluorophores creates a very high signal to noise ratios and minimizes damaging effects of photobleaching. Being a widefield technique, TIRF also allows faster image acquisition than most scanning based confocal setups. At first glance, the low penetration depth of TIRF seems to be incompatible with imaging of bacterial and fungal cells, which are often surrounded by thick cell walls. On the contrary, we have found that the cell walls of yeast and bacterial cells actually improve the usability of TIRF and increase the range of observable structures. Many cellular processes can therefore be directly accessed by TIRF in small, single-cell microorganisms, which often offer powerful genetic manipulation techniques. This allows us to perform in vivo biochemistry experiments, where kinetics of protein interactions and activities can be directly assessed in living cells. We describe here the individual steps required to obtain high quality TIRF images for Saccharomyces cerevisiae or Bacillus subtilis cells. We point out various problems that can affect TIRF visualization of fluorescent probes in cells and illustrate the procedure with several application examples. Finally, we demonstrate how TIRF images can be further improved using established image restoration techniques.

Patchwork organization of the yeast plasma membrane into numerous coexisting domains.

The plasma membrane is made up of lipids and proteins, and serves as an active interface between the cell and its environment. Many plasma-membrane proteins are laterally segregated in the plane of the membrane, but the underlying mechanisms remain controversial. Here we investigate the distribution and dynamics of a representative set of plasma-membrane-associated proteins in yeast cells. These proteins were distributed non-homogeneously in patterns ranging from distinct patches to nearly continuous networks, and these patterns were in turn strongly influenced by the lipid composition of the plasma membrane. Most proteins segregated into distinct domains. However, proteins with similar or identical transmembrane sequences (TMSs) showed a marked tendency to co-localize. Indeed we could predictably relocate proteins by swapping their TMSs. Finally, we found that the domain association of plasma-membrane proteins has an impact on their function. Our results are consistent with self-organization of biological membranes into a patchwork of coexisting domains.