Structure brings clarity: structured illumination microscopy in cell biology.

Biological samples are three dimensional and, therefore, optical sectioning is mandatory for microscopic images to precisely show the localization or function of structures within biological samples. Today, researchers can choose from a variety of methods to obtain optical sections. This article focuses on structured illumination microscopy, which is a group of techniques utilizing a combination of optics and mathematics to obtain optical sections: A structure is imaged onto the sample by optical means and the additional information thereby encoded in the image is used to calculate an optical section from several acquired images. Different methods of structured illumination microscopy (mainly grid projection and aperture correlation) are discussed from a practical point of view, concentrating on advantages, limitations and future prospects of these techniques and their use in cell biology. Structured illumination can also be used to obtain super-resolution information if structures of higher frequency are projected onto the sample. This promising approach to super-resolution microscopy is also briefly discussed from a user's perspective.

Reggies/flotillins regulate cytoskeletal remodeling during neuronal differentiation via CAP/ponsin and Rho GTPases.

The reggies/flotillins were discovered as proteins upregulated during axon regeneration. Here, we show that expression of a trans-negative reggie-1/flotillin-2 deletion mutant, R1EA, which interferes with oligomerization of the reggies/flotillins, inhibited insulin-like growth factor (IGF)-induced neurite outgrowth in N2a neuroblastoma cells and impaired in vitro differentiation of primary rat hippocampal neurons. Cells expressing R1EA formed only short and broad membrane protrusions often with abnormally large growth cones. R1EA expression strongly perturbed the balanced activation of the Rho-family GTPases Rac1 and cdc42. Furthermore, focal adhesion kinase (FAK) activity was also enhanced by R1EA expression, while other signaling pathways like ERK1/2, PKC or PKB signaling were unaffected. These severe signaling defects were caused by an impaired recruitment of the reggie/flotillin-associated adaptor molecule CAP/ponsin to focal contacts at the plasma membrane. Thus, the reggies/flotillins are crucial for coordinated assembly of signaling complexes regulating cytoskeletal remodeling.

Trafficking of the microdomain scaffolding protein reggie-1/flotillin-2.

The reggie/flotillin proteins oligomerize and associate into clusters which form scaffolds for membrane microdomains. Besides their localization at the plasma membrane, the reggies/flotillins reside at various intracellular compartments; however, the trafficking pathways used by reggie-1/flotillin-2 remain unclear. Here, we show that trafficking of reggie-1/flotillin-2 is BFA sensitive and that deletion mutants of reggie-1/flotillin-2 accumulate in the Golgi complex in HeLa, Jurkat and PC12 cells, suggesting Golgi-dependent trafficking of reggie-1/flotillin-2. Using total internal reflection fluorescence microscopy, we observed fast cycling of reggie-1/flotillin-2-positive vesicles at the plasma membrane, which engaged in transient interactions with the plasma membrane only. Reggie-1/flotillin-2 cycling was independent of clathrin, but was inhibited by cholesterol depletion and microtubule disruption. Cycling of reggie-1/flotillin-2 was negatively correlated with cell-cell contact formation but was stimulated by serum, epidermal growth factor and by cholesterol loading mediated by low density lipoproteins. However, reggie-1/flotillin-2 was neither involved in endocytosis of the epidermal growth factor itself nor in endocytosis of GPI-GFPs or the GPI-anchored cellular prion protein (PrP(c)). Reggie-2/flotillin-1 and stomatin-1 also exhibited cycling at the plasma membrane similar to reggie-1/flotillin-2, but these vesicles and microdomains only partially co-localized with reggie-2/flotillin-1. Thus, regulated vesicular cycling might be a general feature of SPFH protein-dependent trafficking.

Linking membrane microdomains to the cytoskeleton: regulation of the lateral mobility of reggie-1/flotillin-2 by interaction with actin.

The reggies/flotillins are oligomeric scaffolding proteins for membrane microdomains. We show here that reggie-1/flotillin-2 microdomains are organized along cortical F-actin in several cell types. Interaction with F-actin is mediated by the SPFH domain as shown by in vivo co-localization and in vitro binding experiments. Reggie-1/flotillin-2 microdomains form independent of actin, but disruption or stabilization of the actin cytoskeleton modulate the lateral mobility of reggie-1/flotillin-2 as shown by FRAP. Furthermore, reggie/flotillin microdomains can efficiently be immobilized by actin polymerisation, while exchange of reggie-1/flotillin-2 molecules between microdomains is enhanced by actin disruption as shown by tracking of individual microdomains using TIRF microscopy.

Preformed reggie/flotillin caps: stable priming platforms for macrodomain assembly in T cells.

T cell activation after contact with an antigen-presenting cell depends on the regulated assembly of the T cell receptor signaling complex, which involves the polarized assembly of a stable, raft-like macrodomain surrounding engaged T cell receptors. Here we show that the preformed reggie/flotillin caps present in resting T cells act as priming platforms for macrodomain assembly. Preformed reggie-1/flotillin-2 caps are exceptionally stable, as shown by fluorescence recovery after photobleaching (FRAP). Upon T cell stimulation, signaling molecules are recruited to the stable reggie/flotillin caps. Importantly, a trans-negative reggie-1/flotillin-2 deletion mutant, which interferes with assembly of the preformed reggie/flotillin cap, impairs raft polarization and macrodomain formation after T cell activation. Accordingly, expression of the trans-negative reggie-1 mutant leads to the incorrect positioning of the guanine nucleotide exchange factor Vav, resulting in defects in cytoskeletal reorganization. Thus, the preformed reggie/flotillin caps are stable priming platforms for the assembly of multiprotein complexes controlling actin reorganization during T cell activation.

Accumulation of FlAsH/Lumio Green in active mitochondria can be reversed by beta-mercaptoethanol for specific staining of tetracysteine-tagged proteins.

Recent advances in the field of small molecule labels for live cell imaging promise to overcome some of the limitations set by the size of fluorescent proteins. We tested the tetracysteine-biarsenical labeling system in live cell fluorescence microscopy of reggie-1/flotillin-2 in HeLa and N2a cells. In both cell types, the biarsenical staining reagent FlAsH/Lumio Green accumulated in active mitochondria and led to mitochondrial swelling. This is indicative of toxic side effects caused by arsenic, which should be considered when this labeling system is to be used in live cell imaging. Mitochondrial accumulation of FlAsH/Lumio Green was reversed by addition of low concentrations of thiol-containing reagents during labeling and a subsequent high stringency thiol wash. Both ethanedithiol and beta-mercaptoethanol proved to be effective. We therefore established a staining protocol using beta-mercaptoethanol as thiol binding site competitor resulting in a specific staining of tetracysteine-tagged reggie-1/flotillin-2 of adequate signal to noise ratio, so that the more toxic and inconvenient ethanedithiol could be avoided. Furthermore, we show that staining efficiency was greatly enhanced by introducing a second tetracysteine sequence in tandem.

Activation of T cell calcium influx by the second messenger ADP-ribose.

Stimulation of Jurkat T cells by high concentrations of concanavalin A (ConA) induced an elevation of the endogenous adenosine diphosphoribose (ADPR) concentration and an inward current significantly different from the Ca2+ release-activated Ca2+ current (I(CRAC)). Electrophysiological characterization and activation of a similar current by infusion of ADPR indicated that the ConA-induced current is carried by TRPM2. Expression of TRPM2 in the plasma membrane of Jurkat T cells was demonstrated by reverse transcription-PCR, Western blot, and immunofluorescence. Inhibition of ADPR formation reduced ConA-mediated, but not store-operated, Ca2+ entry and prevented ConA-induced cell death of Jurkat cells. Moreover, gene silencing of TRPM2 abolished the ADPR- and ConA-mediated inward current. Thus, ADPR is a novel second messenger significantly involved in ConA-mediated cell death in T cells.

PrPc capping in T cells promotes its association with the lipid raft proteins reggie-1 and reggie-2 and leads to signal transduction.

The cellular prion protein (PrPc) resides in lipid rafts, yet the type of raft and the physiological function of PrPc are unclear. We show here that cross-linking of PrPc with specific antibodies leads to 1) PrPc capping in Jurkat and human peripheral blood T cells; 2) to cocapping with the intracellular lipid raft proteins reggie-1 and reggie-2; 3) to signal transduction as seen by MAP kinase phosphorylation and an elevation of the intracellular Ca2+ concentration; 4) to the recruitment of Thy-1, TCR/CD3, fyn, lck and LAT into the cap along with local tyrosine phosphorylation and F-actin polymerization, and later, internalization of PrPc together with the reggies into limp-2 positive lysosomes. Thus, PrPc association with reggie rafts triggers distinct transmembrane signal transduction events in T cells that promote the focal concentration of PrPc itself by guiding activated PrPc into preformed reggie caps and then to the recruitment of important interacting signaling molecules.

Ca2+ release via ryanodine receptors and Ca2+ entry: major mechanisms in NAADP-mediated Ca2+ signaling in T-lymphocytes.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing nucleotide essentially involved in T cell activation. Using combined microinjection and single cell calcium imaging, we demonstrate that co-injection of NAADP and the D-myo-inositol 1,4,5-trisphosphate antagonist heparin did not inhibit Ca2+ mobilization. In contrast, co-injection of the ryanodine receptor antagonist ruthenium red efficiently blocked NAADP induced Ca2+ signalling. This pharmacological approach was confirmed using T cell clones stably transfected with plasmids expressing antisense mRNA targeted specifically against ryanodine receptors. NAADP induced Ca2+ signaling was strongly reduced in these clones. In addition, inhibition of Ca2+ entry by SK&F 96365 resulted in a dramatically decreased Ca2+ signal upon NAADP injection. Gd3+, a known blocker of Ca2+ release activated Ca2+ entry, only partially inhibited NAADP mediated Ca2+ signaling. These data indicate that in T cells (i) ryanodine receptor are the major intracellular Ca2+ release channels involved in NAADP induced Ca2+ signals, and that (ii) such Ca2+ release events are largely amplified by Ca2+ entry.

Amplification and propagation of pacemaker Ca2+ signals by cyclic ADP-ribose and the type 3 ryanodine receptor in T cells.

Ligation of the T-cell receptor/CD3 complex results in global Ca(2+) signals that are essential for T-cell activation. We have recently reported that these global Ca(2+) signals are preceded by localized pacemaker Ca(2+) signals. Here, we demonstrate for the first time for human T cells that an increase in signal frequency of subcellular pacemaker Ca(2+) signals at sites close to the plasma membrane, in the cytosol and in the nucleus depends on the type 3 ryanodine receptor (RyR) and its modulation by cyclic ADP-ribose. The spatial distribution of D-myo-inositol 1,4,5-trisphosphate receptors and RyRs indicates a concerted action of both of these receptors/Ca(2+) channels in the generation of initial pacemaker signals localized close to the plasma membrane. Inhibition or knockdown of RyRs resulted in significant decreases in (1) the frequency of initial pacemaker signals localized close to the plasma membrane, and (2) the frequency of localized pacemaker Ca(2+) signals in the inner cytosol. Moreover, upon microinjection of cyclic ADP-ribose or upon extracellular addition of its novel membrane-permeant mimic N-1-ethoxymethyl-substituted cyclic inosine diphosphoribose, similarly decreased Ca(2+) signals were observed in both type 3 RyR-knockdown cells and in control cells microinjected with the RyR antagonist Ruthenium Red. Taken together, our results show that, under physiological conditions in human T cells, RyRs play crucial roles in the local amplification and the spatiotemporal development of subcellular Ca(2+) pacemaker signals.