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

Lasker, Keren; Diezmann, Alex von; Ahrens, Daniel; Mann, Thomas H.; Moerner, W. E.; Shapiro, Lucy

doi:10.1101/220293

Cited by 13 publications

(14 citation statements)

References 125 publications

(246 reference statements)

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“…Additionally, oligomerization of CckA is critical for DivL stimulation of kinase activity but not its stimulation of CckA phosphatase activity. While we must keep in mind that this reconstitution approach represents a simplified system that lacks additional factors present in vivo , our previous experiments using reconstituted CckA on liposomes have been supported by subsequent work demonstrating agreement that the CckA surface density that is critical for kinase activity in our in vitro experiments matches the surface density of CckA present at the new cell pole in vivo (22). In this study, we reconstitute an additional aspect of subcellular regulation of CckA.…”

Section: Introductionsupporting

confidence: 64%

Integration of cell cycle signals by multi-PAS domain kinases

Mann

Shapiro

2018

Preprint

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20Spatial control of intracellular signaling relies on signaling proteins sensing their 21 subcellular environment. In many cases, a large number of upstream signals are funneled to a 22 master regulator of cellular behavior, but it remains unclear how individual proteins can rapidly 23 integrate a complex array of signals within the appropriate spatial niche within the cell. As a 24 model for how subcellular spatial information can control signaling activity, we have 25 reconstituted the cell pole-specific control of the master regulator kinase/phosphatase CckA from 26 the asymmetrically dividing bacterium Caulobacter crescentus. CckA is active as a kinase only 27 when it accumulates within a microdomain at the new cell pole, where it co-localizes with the 28 pseudokinase DivL. Both proteins contain multiple PAS domains, a multifunctional class of 29 sensory domains present across the kingdoms of life. Here, we show that CckA uses its PAS 30 domains to integrate information from DivL and on its own oligomerization state to control the 31 balance of its kinase and phosphatase activities. We reconstituted the DivL-CckA complex on 32 liposomes in vitro and found that DivL directly controls the CckA kinase-phosphatase switch, 33and that stimulation of either CckA catalytic activity depends on the second of its two PAS 34 domains. We further show that CckA oligomerizes through a multi-domain interaction that is 35 critical for stimulation of kinase activity by DivL, while DivL stimulation of CckA phosphatase 36 activity is independent of CckA homo-oligomerization. Our results broadly demonstrate how 37 signaling factors can leverage information from their subcellular niche to drive spatiotemporal 38 control of cell signaling. 39 40 41 42 Significance 43Cells must constantly make decisions involving many pieces of information at a 44 molecular level. Kinases containing multiple PAS sensory domains detect multiple signals to 45 determine their signaling outputs. In the asymmetrically dividing bacterium Caulobacter 46 crescentus, the multi-sensor proteins DivL and CckA promote different cell types depending 47 upon their subcellular location. We reconstituted the DivL-CckA interaction in vitro and showed 48 that specific PAS domains of each protein function to switch CckA between kinase and 49 phosphatase activities, which reflects their functions in vivo. Within the context of the cell, our 50 reconstitution illustrates how multi-sensor proteins can use their subcellular location to regulate 51 their signaling functions. 52 53

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Section: Introductionsupporting

confidence: 64%

Integration of cell cycle signals by multi-PAS domain kinases

Mann

Shapiro

2018

Preprint

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“…While BR-bodies and CcmM-Rubisco condensates are currently the only bacterial biomolecular condensates whose components have been directly shown to form liquid-like droplets in physiological conditions (Al-Husini et al, 2018; Wang et al, 2019), the cell division protein FtsZ and its inhibitor SlmA were shown to form condensates upon addition of crowding reagents (Monterroso et al, 2019). In addition, other bacterial proteins such as the polar protein scaffold PopZ (Lasker et al, 2018; Zhao et al, 2018) and the nucleoid associated protein DPS (Janissen et al, 2018) have been shown to act as selectively permeable scaffolds, yet biomolecular condensation has not been directly observed.…”

Section: Discussionmentioning

confidence: 99%

BR-bodies provide selectively permeable condensates that stimulate mRNA decay and prevent release of decay intermediates

Al-Husini

Tomares

Pfaffenberger

et al. 2019

Preprint

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AbstractBiomolecular condensates play a key role in organizing RNAs and proteins into membraneless organelles. Bacterial RNP-bodies (BR-bodies) are a biomolecular condensate containing the RNA degradosome mRNA decay machinery, but the biochemical function of such organization remains poorly defined. Here we define the RNA substrates of BR-bodies through enrichment of the bodies followed by RNA-seq. We find that long, poorly translated mRNAs, small RNAs, and antisense RNAs are the main substrates, while rRNA, tRNA, and other conserved ncRNAs are excluded from these bodies. BR-bodies stimulate the mRNA decay rate of enriched mRNAs, helping to reshape the cellular mRNA pool. We also observe that BR-body formation promotes complete mRNA decay, avoiding the build-up of toxic endo-cleaved mRNA decay intermediates. The combined selective permeability of BR-bodies for both, enzymes and substrates together with the stimulation of the sub-steps of mRNA decay provide an effective organization strategy for bacterial mRNA decay.

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“…Tracking CysRsaA anchored to the LPS outer membrane while utilizing a double helix point-spread function for 3D localization 18 allowed calculation of an apparent diffusion coefficient, D=0.077 µm 2 /s (Figure 4e, Methods). A comparison with the recent SMT of a similarly sized transmembrane signaling protein anchored to the inner membrane of C. crescentus , CckA (D=0.0082 µm 2 /s) 32 , reveals that CysRsaA diffuses an order of magnitude faster.…”

Section: Main Textmentioning

confidence: 93%

Continuous, Topologically Guided Protein Crystallization Controls Bacterial Surface Layer Self-Assembly

Comerci

Herrmann

Yoon

et al. 2019

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19Bacteria assemble the cell envelope using localized enzymes to account for growth and 20 division of a topologically complicated surface 1-3 . However, a regulatory pathway has not been 21 identified for assembly and maintenance of the surface layer (S-layer), a 2D crystalline protein 22 coat surrounding the curved 3D surface of a variety of bacteria 4,5 . By specifically labeling, 23 performed time-resolved, super-resolution fluorescence imaging and single-molecule tracking 47 (SMT) of S-layer assembly on living C. crescentus cells. In C. crescentus, the S-layer is made of 48 a single 98 kDa SLP, RsaA, which accounts for around 30% of the cell's total protein synthesis 7 . 49RsaA, like other SLPs, self-assembles into crystalline sheets upon the addition of divalent 50 calcium (Ca 2+ ) in vitro [8][9][10][11][12] . Given the many fitness-related functions ascribed to crystalline 51 bacterial S-layers, we hypothesized that SLP self-assembly may play a role in generating the S-52 layer coat in vivo 4,6 . 53RsaA covers the cellular surface of C. crescentus by forming a 22 nm-repeat hexameric 54 crystal lattice and is non-covalently anchored to an ~18 nm thick LPS outer membrane 13-17 55 (Figure 1a,b). The surface topology of stalked and predivisional C. crescentus includes a 56 cylindrical stalk measuring roughly 100 nm in diameter while the crescentoid cell body 57 approaches 800 nm in width 18,19 (Figure 1d). This large variety of curved topologies guarantees 58 that crystal distortion and defects within the S-layer lattice structure are present, defects which 59 enable complete coverage of the bacterial surface 4,13,20,21 . We can use Gaussian curvature, the 60 product of the maximum and minimum curvatures at a given point, to quantify the cellular 61 topology 22 . Crystalline defects cluster at regions with high absolute values of Gaussian curvature 62 such as the cell poles and division plane, while grain boundaries occur where Gaussian curvature 63 approaches zero such as the cell body (Figure 1d) 20,21 . 64Specifically labeling the S-layer in vivo has proven difficult due to the SLP's life cycle 65 and functions, which include secretion, refolding, anchoring, and crystallization 11,16,17,23,24 . 66

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

Cited by 13 publications

References 125 publications

Integration of cell cycle signals by multi-PAS domain kinases

Integration of cell cycle signals by multi-PAS domain kinases

BR-bodies provide selectively permeable condensates that stimulate mRNA decay and prevent release of decay intermediates

Continuous, Topologically Guided Protein Crystallization Controls Bacterial Surface Layer Self-Assembly

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