FLIRT: fast local infrared thermogenetics for subcellular control of protein function.

FLIRT (fast local infrared thermogenetics) is a microscopy-based technology to locally and reversibly manipulate protein function while simultaneously monitoring the effects in vivo. FLIRT locally inactivates fast-acting temperature-sensitive mutant proteins. We demonstrate that FLIRT can control temperature-sensitive proteins required for cell division, Delta-Notch cell fate signaling, and germline structure in Caenorhabditis elegans with cell-specific and even subcellular precision.

Low Efficiency Upconversion Nanoparticles for High-Resolution Coalignment of Near-Infrared and Visible Light Paths on a Light Microscope.

The combination of near-infrared (NIR) and visible wavelengths in light microscopy for biological studies is increasingly common. For example, many fields of biology are developing the use of NIR for optogenetics, in which an NIR laser induces a change in gene expression and/or protein function. One major technical barrier in working with both NIR and visible light on an optical microscope is obtaining their precise coalignment at the imaging plane position. Photon upconverting particles (UCPs) can bridge this gap as they are excited by NIR light but emit in the visible range via an anti-Stokes luminescence mechanism. Here, two different UCPs have been identified, high-efficiency micro540-UCPs and lower efficiency nano545-UCPs, that respond to NIR light and emit visible light with high photostability even at very high NIR power densities (>25 000 Suns). Both of these UCPs can be rapidly and reversibly excited by visible and NIR light and emit light at visible wavelengths detectable with standard emission settings used for Green Fluorescent Protein (GFP), a commonly used genetically encoded fluorophore. However, the high efficiency micro540-UCPs were suboptimal for NIR and visible light coalignment, due to their larger size and spatial broadening from particle-to-particle energy transfer consistent with a long-lived excited state and saturated power dependence. In contrast, the lower efficiency nano-UCPs were superior for precise coalignment of the NIR beam with the visible light path (∼2 µm versus ∼8 µm beam broadening, respectively) consistent with limited particle-to-particle energy transfer, superlinear power dependence for emission, and much smaller particle size. Furthermore, the nano-UCPs were superior to a traditional two-camera method for NIR and visible light path alignment in an in vivo Infrared-Laser-Evoked Gene Operator (IR-LEGO) optogenetics assay in the budding yeast Saccharomyces cerevisiae. In summary, nano-UCPs are powerful new tools for coaligning NIR and visible light paths on a light microscope.

Functional genomics identifies a requirement of pre-mRNA splicing factors for sister chromatid cohesion.

Sister chromatid cohesion mediated by the cohesin complex is essential for chromosome segregation during cell division. Using functional genomic screening, we identify a set of 26 pre-mRNA splicing factors that are required for sister chromatid cohesion in human cells. Loss of spliceosome subunits increases the dissociation rate of cohesin from chromatin and abrogates cohesion after DNA replication, ultimately causing mitotic catastrophe. Depletion of splicing factors causes defective processing of the pre-mRNA encoding sororin, a factor required for the stable association of cohesin with chromatin, and an associated reduction of sororin protein level. Expression of an intronless version of sororin and depletion of the cohesin release protein WAPL suppress the cohesion defect in cells lacking splicing factors. We propose that spliceosome components contribute to sister chromatid cohesion and mitotic chromosome segregation through splicing of sororin pre-mRNA. Our results highlight the loss of cohesion as an early cellular consequence of compromised splicing. This may have clinical implications because SF3B1, a splicing factor that we identify to be essential for cohesion, is recurrently mutated in chronic lymphocytic leukaemia.

Centralspindlin links the mitotic spindle to the plasma membrane during cytokinesis.

At the end of cell division, cytokinesis splits the cytoplasm of nascent daughter cells and partitions segregated sister genomes. To coordinate cell division with chromosome segregation, the mitotic spindle controls cytokinetic events at the cell envelope. The spindle midzone stimulates the actomyosin-driven contraction of the cleavage furrow, which proceeds until the formation of a microtubule-rich intercellular bridge with the midbody at its centre. The midbody directs the final membrane abscission reaction and has been proposed to attach the cleavage furrow to the intercellular bridge. How the mitotic spindle is connected to the plasma membrane during cytokinesis is not understood. Here we identify a plasma membrane tethering activity in the centralspindlin protein complex, a conserved component of the spindle midzone and midbody. We demonstrate that the C1 domain of the centralspindlin subunit MgcRacGAP associates with the plasma membrane by interacting with polyanionic phosphoinositide lipids. Using X-ray crystallography we determine the structure of this atypical C1 domain. Mutations in the hydrophobic cap and in basic residues of the C1 domain of MgcRacGAP prevent association of the protein with the plasma membrane, and abrogate cytokinesis in human and chicken cells. Artificial membrane tethering of centralspindlin restores cell division in the absence of the C1 domain of MgcRacGAP. Although C1 domain function is dispensable for the formation of the midzone and midbody, it promotes contractility and is required for the attachment of the plasma membrane to the midbody, a long-postulated function of this organelle. Our analysis suggests that centralspindlin links the mitotic spindle to the plasma membrane to secure the final cut during cytokinesis in animal cells.

Mitotic Golgi vesiculation involves mechanisms independent of Ser25 phosphorylation of GM130.

During mitosis, the Golgi undergoes two sequential fragmentation steps to break from ribbon to individual stacks, then from stacks to vesicles. While the mechanism that regulates the first step has been studied, it remains obscure how the second vesiculation step is regulated. It has been suggested that Cdk1-dependent phosphorylation of the cis-Golgi matrix protein GM130 regulates the second step. Here we have tested if phorphorylation of GM130 by Cdk1 is required for Golgi vesiculation and mitotic progression. Inhibition of Cdk1 activity caused a failure of Golgi vesiculation and defects in chromosome congression/segregation. Expression of non-phosphorylatable mutant of GM130 (GM130S25A) in cells depleted of endogenous GM130 caused no apparent defects in Golgi vesiculation and mitotic progression. Similarly, no apparent defects in Golgi vesiculation and mitotic progression were observed when GM130S25A was expressed in GM130-deficient CHO cells. Our observations suggest that while Cdk1 based phosphorylation is essential for mitotic Golgi vesiculation, mammalian cells could possess redundant, S25 phosphorylation of GM130 independent pathways that ensure Golgi vesiculation and mitotic progression.