T cell antigen recognition requires T cell antigen receptors (TCRs) engaging MHC-embedded antigenic peptides (pMHCs) within the contact region of a T cell with its conjugated antigen-presenting cell. Despite micromolar TCR:pMHC affinities, T cells respond to even a single antigenic pMHC, and higher-order TCRs have been postulated to maintain high antigen sensitivity and trigger signaling. We interrogated the stoichiometry of TCRs and their associated CD3 subunits on the surface of living T cells through single-molecule brightness and single-molecule coincidence analysis, photon-antibunching-based fluorescence correlation spectroscopy and Förster resonance energy transfer measurements. We found exclusively monomeric TCR-CD3 complexes driving the recognition of antigenic pMHCs, which underscores the exceptional capacity of single TCR-CD3 complexes to elicit robust intracellular signaling.

The generation of mature mRNAs from most genes (about 80%-90%) in autotrophic eukaryotes requires the removal of noncoding sequences (introns) and splicing of the coding regions (exons; Labadorf et al., 2010). During splicing in some precursor messenger RNAs (pre-mRNAs), the same splice sites are always used, referred to as constitutive splicing (CS), resulting in a single transcript from a gene. However, from many pre-mRNAs, multiple mature mRNAs are generated from a single gene by alternative splicing (AS), where different combinations of splice sites are used. Both CS and AS are critical to the proper expression of intron-containing genes. Recent transcriptome-wide analysis of AS using high-throughput sequencing indicates that pre-mRNAs from up to 42% of introncontaining genes in Arabidopsis (Arabidopsis thaliana; Filichkin et al., 2010) and about 48% in rice (Oryza sativa; Lu et al., 2010) are alternatively spliced, whereas about 95% of human genes are alternatively spliced (Pan et al., 2008). In addition to pre-mRNAs, some primary microRNAs (pri-miRNAs) are also subject to CS and AS (Hirsch et al., 2006;Szarzynska et al., 2009;Mica et al., 2010). AS increases the protein-coding capacity of a genome and generates functionally different proteins from the same gene (Reddy, 2007). AS results in protein isoforms with loss or gain of function and altered subcellular localization, protein stability, and/or posttranslational modifications. Furthermore, AS plays an important role in gene regulation through regulated unproductive splicing and translation, leading to RNA degradation by mRNA surveillance mechanisms, differential recruitment of mRNAs to ribosomes, or translatability of splice variants (Kurihara et al., 2009;Licatalosi and Darnell, 2010;Palusa and Reddy, 2010). Numerous spliceosomal proteins either promote or suppress splicing by interacting with splicing regulatory elements on the pre-mRNAs. In recent years, the localization and dynamics of some splicing regulators in plants have been analyzed using a variety of approaches. This review summarizes the current status of research in this area, with emphasis on RNAbinding proteins (RBPs) that are involved in splicing regulation, discusses open questions, and presents some approaches to address these questions. SPLICING REGULATORSpre-mRNAs splicing takes place cotranscriptionally in the spliceosome, a large multicomponent complex composed of small nuclear RNAs (snRNAs) and about 170 proteins, many of which are involved in the regulation of splicing (Wahl et al., 2009;Valadkhan and Jaladat, 2010). The composition of the human and yeast spliceosome has been analyzed in great detail (Wahl et al., 2009); however, information on plant splicing has been scarce, as no in vitro assembly of a functional plant spliceosome has been possible to date. A detailed search for Arabidopsis orthologs of the human spliceosome proteome revealed that most splicing factors are present in the Arabidopsis genome, indicating a similar complexity (Barta et al., 2011). Interestingly, m...