The RootScope: A Simple High-Throughput Screening System For Quantitating Gene Expression Dynamics In Plant Roots

Background: High temperature stress responses are vital for plant survival. The mechanisms that plants use to sense high temperatures are only partially understood and involve multiple sensing and signaling pathways. Here we describe the development of the RootScope, an automated microscopy system for quantitating heat shock responses in plant roots.Results: The promoter of Hsp17.6 was used to build a Hsp17.6(p):GFP transcriptional reporter that is induced by heat shock in Arabidopsis. An automated fluorescence microscopy system which enables multiple roots to be imaged in rapid succession was used to quantitate Hsp17.6p: GFP response dynamics. Hsp17.6(p):GFP signal increased with temperature increases from 28 degrees C to 37 degrees C. At 40 degrees C the kinetics and localization of the response are markedly different from those at 37 degrees C. This suggests that different mechanisms mediate heat shock responses above and below 37 degrees C. Finally, we demonstrate that Hsp17.6(p):GFP expression exhibits wave like dynamics in growing roots.Conclusions: The RootScope system is a simple and powerful platform for investigating the heat shock response in plants

Communications Biophysics

Contains reports on nine research projects split into four sections.National Institutes of Health (Grant 5 P01 NS13126)National Institutes of Health (Grant 5 K04 NS00113)National Institutes of Health (Training Grant 5 T32 NS07047)National Institutes of Health (Grant 5 ROl NS11153-03)National Institutes of Health (Fellowship 1 T32 NS07099-01)National Science Foundation (Grant BNS77-16861)National Institutes of Health (Grant 5 ROl NS10916)National Institutes of Health (Grant 5 ROl NS12846)National Science Foundation (Grant BNS77-21751)National Institutes of Health (Grant 1 RO1 NS14092)Health Sciences FundNational Institutes of Health (Grant 2 R01 NS11680)National Institutes of Health (Grant 2 RO1 NS11080)National Institutes of Health (Training Grant 5 T32 GM07301

Communication Biophysics

Contains reports on six research projects.National Institutes of Health (Grant 5 PO1 NS13126)National Institutes of Health (Grant 5 RO1 NS18682)National Institutes of Health (Grant 5 RO1 NS20322)National Institutes of Health (Grant 5 R01 NS20269)National Institutes of Health (Grant 5 T32NS 07047)Symbion, Inc.National Science Foundation (Grant BNS 83-19874)National Science Foundation (Grant BNS 83-19887)National Institutes of Health (Grant 6 RO1 NS 12846)National Institutes of Health (Grant 1 RO1 NS 21322

Mechanisms of mTORC1 signal regulation by the Rag GTPases

The decision of whether to allocate resources toward cellular growth or toward quality control is a matter of cellular life and death; disruption of growth pathways is an emerging driving force in diseases ranging from cancer to neurodegeneration. In mammalian cells, the protein kinase activity of mTORC1 promotes cellular anabolism and impedes cellular catabolism, ultimately achieving a balance that dictates the rate of cell growth. In response to nutrient levels, mTORC1 is activated upon recruitment to the lysosome, an organelle whose role as a nutrient sensing integrator has recently come into focus. The Rag GTPases are required for mTORC1 recruitment to the lysosome, but the mechanisms via which the Rags sense nutrients and precisely couple the degree of mTORC1 lysosomal recruitment levels to the level of available nutrients were unknown.I report new live-imaging and reconstitution approaches that enabled the discovery that when the Rags transition from their inactive nucleotide binding state to their active nucleotide state in response to nutrient stimulation, they also loosen their binding affinity for their lysosomal scaffold, Ragulator. The resulting spatial cycling between the lysosome and the cytoplasm ultimately limits mTORC1 accumulation on its Rag-Ragulator lysosomal scaffold, and promotes rapid responsiveness of mTORC1.Next, I asked whether the nucleotide states of the two Rag GTPase domains are coordinated. Prior work had established that a complex of Folliculin (FLCN) and FLCN-interacting protein 2 (FLCN:FNIP2) serves as a RagC-specific GTPase-activating protein (GAP) and thus has a positive role in mTORC1 stimulation. However, genetic evidence placed FLCN as a tumor suppressor, suggesting a negative role. I reconstituted the “Lysosomal Folliculin Complex” (LFC), a supercomplex composed of Ragulator, inactive-loaded Rags, and FLCN:FNIP2 that localizes to lysosomes. I discovered that in the LFC, FLCN:FNIP2 clamps Rags in their inactive state (RagAGDP:RagCGTP) by directly inhibiting nucleotide exchange in RagA, concomitant with inhibition of its RagC GAP activity, a conclusion reinforced by a high-resolution (3.6 Å) structure of the LFC. Thus, when nutrients are low, FLCN:FNIP2 is able to maintain the Rag heterodimer in its inactive state, but, in response to a rise in nutrients, FLCN:FNIP2 is converted into a functional GAP. Finally, by assessing newly available structures of active nucleotide-bound and inactive nucleotide-bound Rag heterodimers, along with recent structural information about Rag interactors, I was able to assemble an integrated structure-guided model of the Rag-mediated cycle of recruitment and activation of mTORC1 at the lysosome. My findings increase our understanding of the molecular logic of nutrient sensing and point to new opportunities for manipulating mTORC1 signaling in disease contexts

Mechanisms of mTORC1 signal regulation by the Rag GTPases

The decision of whether to allocate resources toward cellular growth or toward quality control is a matter of cellular life and death; disruption of growth pathways is an emerging driving force in diseases ranging from cancer to neurodegeneration. In mammalian cells, the protein kinase activity of mTORC1 promotes cellular anabolism and impedes cellular catabolism, ultimately achieving a balance that dictates the rate of cell growth. In response to nutrient levels, mTORC1 is activated upon recruitment to the lysosome, an organelle whose role as a nutrient sensing integrator has recently come into focus. The Rag GTPases are required for mTORC1 recruitment to the lysosome, but the mechanisms via which the Rags sense nutrients and precisely couple the degree of mTORC1 lysosomal recruitment levels to the level of available nutrients were unknown.I report new live-imaging and reconstitution approaches that enabled the discovery that when the Rags transition from their inactive nucleotide binding state to their active nucleotide state in response to nutrient stimulation, they also loosen their binding affinity for their lysosomal scaffold, Ragulator. The resulting spatial cycling between the lysosome and the cytoplasm ultimately limits mTORC1 accumulation on its Rag-Ragulator lysosomal scaffold, and promotes rapid responsiveness of mTORC1.Next, I asked whether the nucleotide states of the two Rag GTPase domains are coordinated. Prior work had established that a complex of Folliculin (FLCN) and FLCN-interacting protein 2 (FLCN:FNIP2) serves as a RagC-specific GTPase-activating protein (GAP) and thus has a positive role in mTORC1 stimulation. However, genetic evidence placed FLCN as a tumor suppressor, suggesting a negative role. I reconstituted the “Lysosomal Folliculin Complex” (LFC), a supercomplex composed of Ragulator, inactive-loaded Rags, and FLCN:FNIP2 that localizes to lysosomes. I discovered that in the LFC, FLCN:FNIP2 clamps Rags in their inactive state (RagAGDP:RagCGTP) by directly inhibiting nucleotide exchange in RagA, concomitant with inhibition of its RagC GAP activity, a conclusion reinforced by a high-resolution (3.6 Å) structure of the LFC. Thus, when nutrients are low, FLCN:FNIP2 is able to maintain the Rag heterodimer in its inactive state, but, in response to a rise in nutrients, FLCN:FNIP2 is converted into a functional GAP. Finally, by assessing newly available structures of active nucleotide-bound and inactive nucleotide-bound Rag heterodimers, along with recent structural information about Rag interactors, I was able to assemble an integrated structure-guided model of the Rag-mediated cycle of recruitment and activation of mTORC1 at the lysosome. My findings increase our understanding of the molecular logic of nutrient sensing and point to new opportunities for manipulating mTORC1 signaling in disease contexts

Quantitative 4D tracking analysis and chemical induction of heat shock granules during cytosolic misfolded protein stress

Heat Shock Granules (HSGs) are subcellular structures composed of small Heat Shock Proteins (sHSPs) and misfolded proteins that form in response to heat stress in plants. While sHSPs are found in other organisms, HSGs have only been reported in plant cells and only in response to heat stress. This thesis examines the signaling pathways that regulate the transcription of sHSPs and the formation of HSGs and investigates whether heat is the only stress that could activate these pathways. By visualizing HSGs in an Arabidopsis thaliana BOBBERl:GFP reporter line using still and 4-D confocal microscopy, we characterize HSG formation and HSG structural qualities such as volume and shape. 4D tracking is used to describe dynamic behavior. We also show that inducing protein misfolding by treating live seedlings with amino acid analog L-Azetidine2-Carboxylic Acid (AZC) or proteasome inhibitor MG132 induces granule formation. We propose that the term Heat Shock Granule is a misnomer, since HSG formation can be catalyzed by misfolded protein stress in the absence of heat treatment

Structural mechanism for amino acid-dependent Rag GTPase nucleotide state switching by SLC38A9.

The Rag GTPases (Rags) recruit mTORC1 to the lysosomal membrane in response to nutrients, where it is then activated in response to energy and growth factor availability. The lysosomal folliculin (FLCN) complex (LFC) consists of the inactive Rag dimer, the pentameric scaffold Ragulator, and the FLCN:FNIP2 (FLCN-interacting protein 2) GTPase activating protein (GAP) complex, and prevents Rag dimer activation during amino acid starvation. How the LFC is disassembled upon amino acid refeeding is an outstanding question. Here we show that the cytoplasmic tail of the human lysosomal solute carrier family 38 member 9 (SLC38A9) destabilizes the LFC and thereby triggers GAP activity of FLCN:FNIP2 toward RagC. We present the cryo-EM structures of Rags in complex with their lysosomal anchor complex Ragulator and the cytoplasmic tail of SLC38A9 in the pre- and post-GTP hydrolysis state of RagC, which explain how SLC38A9 destabilizes the LFC and so promotes Rag dimer activation

A point mutation in the nucleotide exchange factor eIF2B constitutively activates the integrated stress response by allosteric modulation.

In eukaryotic cells, stressors reprogram the cellular proteome by activating the integrated stress response (ISR). In its canonical form, stress-sensing kinases phosphorylate the eukaryotic translation initiation factor eIF2 (eIF2-P), which ultimately leads to reduced levels of ternary complex required for initiation of mRNA translation. Previously we showed that translational control is primarily exerted through a conformational switch in eIF2's nucleotide exchange factor, eIF2B, which shifts from its active A-State conformation to its inhibited I-State conformation upon eIF2-P binding, resulting in reduced nucleotide exchange on eIF2 (Schoof et al. 2021). Here, we show functionally and structurally how a single histidine to aspartate point mutation in eIF2B's β subunit (H160D) mimics the effects of eIF2-P binding by promoting an I-State like conformation, resulting in eIF2-P independent activation of the ISR. These findings corroborate our previously proposed A/I-State model of allosteric ISR regulation