Biology subjects

Shapiro, L.

Publications and source records attributed to Shapiro, L..

6 recordsLinked to original sources

bioRxiv2018

Integration of cell cycle signals by multi-PAS domain kinases

Spatial control of intracellular signaling relies on signaling proteins sensing their subcellular environment. In many cases, a large number of upstream signals are funneled to a master regulator of cellular behavior, but it remains unclear how individual proteins can rapidly integrate a complex array of signals within the appropriate spatial niche within the cell. As a model for how subcellular spatial information can control signaling activity, we have reconstituted the cell pole-specific control of the master regulator kinase/phosphatase CckA from the asymmetrically dividing bacterium Caulobacter crescentus. CckA is active as a kinase only when it accumulates within a microdomain at the new cell pole, where it co-localizes with the pseudokinase DivL. Both proteins contain multiple PAS domains, a multifunctional class of sensory domains present across the kingdoms of life. Here, we show that CckA uses its PAS domains to integrate information from DivL and on its own oligomerization state to control the balance of its kinase and phosphatase activities. We reconstituted the DivL-CckA complex on liposomes in vitro and found that DivL directly controls the CckA kinase-phosphatase switch, and that stimulation of either CckA catalytic activity depends on the second of its two PAS domains. We further show that CckA oligomerizes through a multi-domain interaction that is critical for stimulation of kinase activity by DivL, while DivL stimulation of CckA phosphatase activity is independent of CckA homo-oligomerization. Our results broadly demonstrate how signaling factors can leverage information from their subcellular niche to drive spatiotemporal control of cell signaling.\n\nSignificanceCells must constantly make decisions involving many pieces of information at a molecular level. Kinases containing multiple PAS sensory domains detect multiple signals to determine their signaling outputs. In the asymmetrically dividing bacterium Caulobacter crescentus, the multi-sensor proteins DivL and CckA promote different cell types depending upon their subcellular location. We reconstituted the DivL-CckA interaction in vitro and showed that specific PAS domains of each protein function to switch CckA between kinase and phosphatase activities, which reflects their functions in vivo. Within the context of the cell, our reconstitution illustrates how multi-sensor proteins can use their subcellular location to regulate their signaling functions.

biochemistry↗

bioRxiv2018

Structural survey of HIV-1-neutralizing antibodies targeting Env trimer delineates epitope categories and suggests vaccine templates

HIV-1 broadly neutralizing antibodies are desired for their therapeutic potential and as templates for vaccine design. Such antibodies target the HIV-1-envelope (Env) trimer, which is shielded from immune recognition by extraordinary glycosylation and sequence variability. Recognition by broadly neutralizing antibodies thus provides insight into how antibody can bypass these immune-evasion mechanisms. Remarkably, antibodies neutralizing >25% of HIV-1 strains have now been identified that recognize all major exposed surfaces of the prefusion-closed Env trimer. Here we analyzed all 206 broadly neutralizing antibody-HIV-1 Env complexes in the PDB with resolution suitable to define their interaction chemistries. These segregated into 20 antibody classes based on ontogeny and recognition, and into 6 epitope categories (V1V2, glycan-V3, CD4-binding site, silent face center, fusion peptide, and subunit interface) based on recognized Env residues. We measured antibody neutralization on a 208-isolate panel and analyzed features of paratope and B cell ontogeny. The number of protruding loops, CDR H3 length, and level of somatic hypermutation for broadly HIV-1 neutralizing antibodies were significantly higher than for a comparison set of non-HIV-1 antibodies. For epitope, the number of independent sequence segments was higher (P < 0.0001), as well as the glycan component surface area (P = 0.0005). Based on B cell ontogeny, paratope, and breadth, the CD4-binding site antibody IOMA appeared to be a promising candidate for lineage-based vaccine design. In terms of epitope-based vaccine design, antibody VRC34.01 had few epitope segments, low epitope-glycan content, and high epitope-conformational variability, which may explain why VRC34.01-based design is yielding promising vaccine results.

bioinformatics↗

bioRxiv2018

Cell cycle-controlled clearance of the CcrM DNA methyltransferase by Lon is dependent on DNA-facilitated proteolysis and substrate polar sequestration

N6-adenine methylation catalyzed by the DNA methyltransferase CcrM is an essential epigenetic event of the Caulobacter cell cycle. Limiting CcrM to a specific time period during the cell cycle relies on temporal control of ccrM transcription and CcrM proteolysis. We investigated how Lon, a protease from AAA+ superfamily conserved from bacteria to humans, temporally degrades CcrM to maintain differential chromosomal methylation state, thereby regulating transcription factor synthesis and enabling cell cycle progression. We demonstrate that CcrM degradation by Lon requires DNA as an adaptor for robust proteolysis. Lon, a DNA-bound protein, is constitutively active throughout the cell cycle, but allows CcrM mediated DNA methylation only when CcrM is transcribed and translated upon completion of DNA replication. An additional mechanism to limit CcrM activity to a narrow window of the cell cycle is its sequestration to the pole of the progeny stalked cell, which prevents physical contact with DNA-bound Lon. Thus, we have provided evidence for a novel mechanism for substrate selection by the Lon protease, providing robust cell cycle control mediated by DNA methylation.

molecular biology↗

bioRxiv2017

Phospho-signal flow from a pole-localized microdomain spatially patterns transcription factor activity

Selective recruitment and concentration of signaling proteins within membrane-less compartments is a ubiquitous mechanism for subcellular organization. However, little is known about how such dynamic recruitment patterns intracellular signaling and cellular development. Here, we combined transcriptional profiling, reaction-diffusion modeling, and single-molecule tracking to study signal exchange in and out of a microdomain at the cell pole of the asymmetrically dividing bacterium Caulobacter crescentus. Our study revealed that the microdomain is selectively permeable, and that each protein in the signaling pathway that activates the cell fate transcription factor CtrA is sequestered and uniformly concentrated within the microdomain or its proximal membrane. Restricted rates of entry into and escape from the microdomain enhance phospho-signaling, leading to a sublinear gradient of CtrA~P along the long axis of the cell. The spatial patterning of CtrA~P creates a gradient of transcriptional activation that serves to prime asymmetric development of the two daughter cells.

cell biology↗

bioRxiv2017

Routine Single Particle CryoEM Sample and Grid Characterization by Tomography

Single particle cryo-electron microscopy (cryoEM) is often performed under the assumption that particles are freely floating away from the air-water interfaces and in thin, vitreous ice. In this study, we performed fiducial-less tomography on over 50 different cryoEM grid/sample preparations to determine the particle distribution within the ice and the overall geometry of the ice in grid holes. Surprisingly, by studying particles in holes in 3D from over 1,000 tomograms, we have determined that the vast majority of particles (approximately 90%) are adsorbed to an air-water interface. The implications of this observation are wide-ranging, with potential ramifications regarding protein denaturation, conformational change, and preferred orientation. We also show that fiducial-less cryo-electron tomography on single particle grids may be used to determine ice thickness, optimal single particle collection areas and strategies, particle heterogeneity, and de novo models for template picking and single particle alignment.

biophysics↗

bioRxiv2017

Spatial organization and dynamics of RNase E and ribosomes in Caulobacter crescentus

We report the dynamic spatial organization of Caulobacter crescentus RNase E (RNA degradosome) and ribosomal protein L1 (ribosome) using 3D single particle tracking and super-resolution microscopy. RNase E formed clusters along the central axis of the cell, while weak clusters of ribosomal protein L1 were deployed throughout the cytoplasm. These results contrast with RNase E and ribosome distribution in E. coli, where RNase E co-localizes with the cytoplasmic membrane and ribosomes accumulate in polar nucleoid-free zones. For both RNase E and ribosomes in Caulobacter, we observed a decrease in confinement and clustering upon transcription inhibition and subsequent depletion of nascent RNA, suggesting that RNA substrate availability for processing, degradation, and translation facilitates confinement and clustering. Moreover, RNase E cluster positions correlate with the subcellular location of chromosomal loci of two highly transcribed ribosomal RNA genes, suggesting that RNase Es function in ribosomal RNA processing occurs at the site of rRNA synthesis. Thus, components of the RNA degradosome and ribosome assembly are spatiotemporally organized in Caulobacter, with chromosomal readout serving as the template for this organization.

biophysics↗