The dynamic behavior of proteins is critical for cellular homeostasis. However, analyzing dynamics of proteins and protein complexes in vivo has been difficult. Here we describe recombination-induced tag exchange (RITE), a genetic method that induces a permanent epitope-tag switch in the coding sequence after a hormone-induced activation of Cre recombinase. The time-controlled tag switch provides a unique ability to detect and separate old and new proteins in time and space, which opens up opportunities to investigate the dynamic behavior of proteins. We validated the technology by determining exchange of endogenous histones in chromatin by biochemical methods and by visualizing and quantifying replacement of old by new proteasomes in single cells by microscopy. RITE is widely applicable and allows probing spatiotemporal changes in protein properties by multiple methods.chromatin | histone | proteasome | protein dynamics | turnover P roteins are dynamic molecules. Their abundance is controlled by synthesis and degradation and they can be subject to posttranslational processing, modification, and demodification. In addition, most proteins are very mobile and undergo interactions with multiple other protein partners (1-4). However, little is known about the dynamics of proteins within macromolecular complexes in vivo (2, 4). Studying time-dependent changes in physical properties of proteins or protein turnover requires methods to distinguish resident (old) proteins from new proteins. Current methods that do so are usually based on fluorescent reporters or differential chemical labeling. For example, fluorescence recovery after photo bleaching relies on exchange of the old bleached protein by nonbleached proteins (1, 3, 4). Alternative methods involve time-dependent changes in fluorescence, nonspecific pulse-chase labeling of proteins with labeled amino acids, or labeling with chemical dyes that specifically bind to short tags (5-7). Although suitable for detection of proteins by microscopy or mass spectrometry, a limitation of these methods is that they do not provide a handle for biochemical analysis of old and new proteins and their complexes. To solve this problem and to eliminate the requirement for chemical labels or UV light we developed recombination-induced tag exchange (RITE), a method in which a genetic epitope tag is switched by transient induction of a site-specific recombinase. As a consequence, old and newly synthesized proteins are differentially tagged, which enables monitoring of protein dynamics by multiple techniques, as illustrated here. In contrast to inducible expression strategies (8-12), differential tagging by a time-controlled site-specific protease (13), or the labeling methods described above, RITE allows parallel detection and purification of old and new proteins under physiological conditions and over long periods of time.We used RITE to probe the stability of chromatin. Photobleaching experiments using histones tagged with fluorescent reporters suggest that chromatin is a static comp...

To facilitate the myriad of different (signaling) processes that take place at the plasma membrane, cells depend on a high degree of membrane protein organization. Important mediators of this organization are tetraspanin proteins. Tetraspanins interact laterally among themselves and with partner proteins to control the spatial organization of membrane proteins in large networks called the tetraspanin web. The molecular interactions underlying the formation of the tetraspanin web were hitherto mainly described based on their resistance to different detergents, a classification which does not necessarily correlate with functionality in the living cell. To look at these interactions from a more physiological point of view, this review discusses tetraspanin interactions based on their function in the tetraspanin web: (1) intramolecular interactions supporting tetraspanin structure, (2) tetraspanin-tetraspanin interactions supporting web formation, (3) tetraspanin-partner interactions adding functional partners to the web and (4) cytosolic tetraspanin interactions regulating intracellular signaling. The recent publication of the first full-length tetraspanin crystal structure sheds new light on both the intra- and intermolecular tetraspanin interactions that shape the tetraspanin web. Furthermore, recent molecular dynamic modeling studies indicate that the binding strength between tetraspanins and between tetraspanins and their partners is the complex sum of both promiscuous and specific interactions. A deeper insight into this complex mixture of interactions is essential to our fundamental understanding of the tetraspanin web and its dynamics which constitute a basic building block of the cell surface.

Following mitosis, daughter cells must inherit a functional set of essential proteins and organelles. We applied a genetic tool to simultaneously monitor the kinetics and distribution of old and new proteins marking all intracellular compartments in budding yeasts. Most organelles followed a general pattern whereby preexisting proteins are symmetrically partitioned followed by template-based incorporation of new proteins. Peroxisomes belong to this group, supporting a model of biogenesis by growth and division from preexisting peroxisomes. We detected two exceptions: the nuclear pore complex (NPC) and the spindle pole body (SPB). Old NPCs are stably inherited during successive generations but remained separated from new NPCs, which are incorporated de novo in mother and daughter cells. Only the SPB displayed asymmetrical distribution, with old components primarily inherited by daughter cells and new proteins equally incorporated in both cells. Our analysis resolves conflicting models (peroxisomes, NPC) and reveals unique patterns (NPC, SPB) of organelle inheritance.Saccharomyces cerevisiae | protein dynamics | nuclear envelope | centrosome | live-cell imaging

Activation of B cells by the binding of antigens to the B cell receptor (BCR) requires the protein kinase C (PKC) family member PKCβ. Because PKCs must translocate to the plasma membrane to become activated, we investigated the mechanisms regulating their spatial distribution in mouse and human B cells. Through live-cell imaging, we showed that BCR-stimulated production of the second messenger diacylglycerol (DAG) resulted in the translocation of PKCβ from the cytosol to plasma membrane regions containing the tetraspanin protein CD53. CD53 was specifically enriched at sites of BCR signaling, suggesting that BCR-dependent PKC signaling was initiated at these tetraspanin microdomains. Fluorescence lifetime imaging microscopy studies confirmed the molecular recruitment of PKC to CD53-containing microdomains, which required the amino terminus of CD53. Furthermore, we showed that -deficient B cells were defective in the phosphorylation of PKC substrates. Consistent with this finding, PKC recruitment to the plasma membrane was impaired in both mouse and human-deficient B cells compared to that in their wild-type counterparts. These data suggest that CD53 promotes BCR-dependent PKC signaling by recruiting PKC to the plasma membrane so that it can phosphorylate its substrates and that tetraspanin-containing microdomains can act as signaling hotspots in the plasma membrane.

Membrane protein organization is essential for proper cellular functioning and the result of a dynamic exchange between protein monomers, nanoscale protein clusters, and microscale higher-order structures. This exchange is affected by both lipid bilayer intrinsic factors, such as lipid rafts and tetraspanins, and extrinsic factors, such as cortical actin and galectins. Because membrane organizers act jointly like instruments in a symphony, it is challenging to define the 'key' organizers. Here, we posit, for the first time, definitions of key intrinsic and extrinsic membrane organizers. Tetraspanin nanodomains are key organizers that are often overlooked. We discuss how different key organizers can collaborate, which is important to get a full grasp of plasma membrane biology. Open Questions in Plasma Membrane Biology HighlightsNovel definitions are postulated for membrane organizers intrinsic and extrinsic to the lipid bilayer.Membrane protein organization is the result of a dynamic exchange between protein monomers, nanoscale protein clusters, and microscale higher-order structures.Tetraspanins are key membrane organizers that should be considered in future plasma membrane studies.Membrane organizers act together in a dynamic manner like instruments playing a symphony.