The spliceosome is the complex macromolecular machine responsible for removing introns from precursors to mRNAs (pre-mRNAs). We combined yeast genetic engineering, chemical biology, and multi-wavelength fluoresence microscopy to follow assembly of single spliceosomes in real time in whole cell extracts. We find that individual spliceosomal subcomplexes associate with pre-mRNA sequentially via an ordered pathway to yield functional spliceosomes, and that association of every subcomplex is reversible. Further, early subcomplex binding events do not fully commit a pre-mRNA to splicing; rather commitment increases as assembly proceeds. These findings have important implications for the regulation of alternative splicing. This experimental strategy should prove widely useful for mechanistic analysis of other macromolecular machines in environments approaching the complexity of living cells.

No abstract

Chemical tags are now viable alternatives to fluorescent proteins for labeling proteins in living cells with organic fluorophores that have improved brightness and other specialized properties. Recently, we successfully rendered our TMP-tag covalent with a proximity-induced reaction between the protein tag and the ligand-fluorophore label. This initial design, however, suffered from slow in vitro labeling kinetics and limited live cell protein labeling. Thus, here we report a second-generation covalent TMP-tag that has a fast labeling half-life and can readily label a variety of intracellular proteins in living cells. Specifically, we designed an acrylamide-trimethoprim-fluorophore (A-TMP-fluorophore v2.0) electrophile with an optimized linker for fast reaction with a cysteine (Cys) nucleophile engineered just outside the TMP-binding pocket of Escherichia coli dihydrofolate reductase (eDHFR) and developed an efficient chemical synthesis for routine production of a variety of A-TMP-probe v2.0 labels. We then screened a panel of eDHFR:Cys variants and identified eDHFR:L28C as having an 8-min half-life for reaction with A-TMP-biotin v2.0 in vitro. Finally, we demonstrated live cell imaging of various cellular protein targets with A-TMP-fluorescein, A-TMP-Dapoxyl, and A-TMP-Atto655. With its robustness, this second-generation covalent TMP-tag adds to the limited number of chemical tags that can be used to covalently label intracellular proteins efficiently in living cells. Moreover, the success of this second-generation design further validates proximity-induced reactivity and organic chemistry as tools not only for chemical tag engineering but also more broadly for synthetic biology.

Chemical tags for live cell imaging are emerging as viable alternatives to the fluorescent proteins for labeling proteins with small molecule probes. Among reported chemical tags, trimethoprim (TMP)-tag stands out for having sufficient cell permeability and selectivity to allow imaging of intracellular proteins. TMP-tag provides a non-covalent label in which the protein of interest is fused to E. coli dihydrofolate reductase (DHFR) and then labeled with a cell permeable TMP-probe heterodimer. To complement the utility of the non-covalent TMP-tag, we sought to render the TMP-tag covalent for applications such as single-molecule tracking and pulse-chase labeling that would benefit from a more permanent modification. Based on the long-standing use of proximity-induced reactivity for irreversible inhibitor design and its more recent application to in vitro chemical biology tools, we designed an eDHFR variant with a unique Cys residue positioned to react with an acrylamide electrophile installed on the TMP-probe label. In vitro experiments show that the eDHFR:Leu28Cys nucleophile reacts rapidly and quantitatively with the TMP-acrylamide-probe. Most significantly, the balance in reactivity provided by the acrylamide electrophile allows intracellular proteins tagged with eDHFR:Leu28Cys to be labeled with a TMP-acrylamide-fluorescein heterotrimer in live cells with minimal background. Thus, the TMP electrophile described here can be used immediately as a covalent chemical tag in live cells. Moreover, proximity-induced reactivity is shown to be sufficiently selective for use in a living cell, suggesting a general approach for the development of orthogonal covalent chemical tags from existing non-covalent ligand-protein pairs.

Monodomain liquid crystal elastomers (LCEs) are new materials uniquely suitable for artificial muscles, as they undergo large reversible uniaxial shape changes, with strains of 20-500% and stresses of 10-100 kPa, falling exactly into the dynamic range of a muscle. LCEs exhibit little to no fatigue over thousands of actuation cycles. Their practical use has been limited, however, owing to the difficulty of synthesizing components, achieving consistent alignment during cross-linking across the whole material and often a high nematic-isotropic phase transition temperature. The most widely studied method for LC alignment involves mechanical stretching of the material during one of two cross-linking steps, which makes fabrication difficult to control and lends itself mainly to samples that can be easily grasped (with sizes of the order of mm). In this article, we describe a method of adapting the LCE synthesis to microscale objects, achieving monodomain alignment with a single cross-linking step, and lowering the cycling temperature. LCE precursor droplets are embedded in and then stretched in a polymer matrix at high temperature. Confinement of the uniaxially stretched droplets maintains the alignment achieved during stretching and allows us to eliminate one of the cross-linking steps and the variability associated with it. Adding a comonomer during the polymerization leads to lowering of the nematic-to-isotropic transition temperature (58 °C), significantly expanding the range of potential applications for these micromuscles. We demonstrate reversible thermal switching of the micromuscles in line with the largest strain changes observed for side-chain LCEs and a differential scanning calorimetry characterization of the material phase transitions. The method demonstrates the parallel fabrication of many microscale actuators and is amenable to further scale-up and manufacturing.