Tonoplast and Peroxisome Targeting of γ-tocopherol N-methyltransferase Homologs Involved in the Synthesis of Monoterpene Indole Alkaloids

Many plant species from the Apocynaceae, Loganiaceae and Rubiaceae families evolved a specialized metabolism leading to the synthesis of a broad palette of monoterpene indole alkaloids (MIAs). These compounds are believed to constitute a cornerstone of the plant chemical arsenal but above all several MIAs display pharmacological properties that have been exploited for decades by humans to treat various diseases. It is established that MIAs are produced in planta due to complex biosynthetic pathways engaging a multitude of specialized enzymes but also a complex tissue and subcellular organization. In this context, N-methyltransferases (NMTs) represent an important family of enzymes indispensable for MIA biosynthesis but their characterization has always remained challenging. In particular, little is known about the subcellular localization of NMTs in MIA-producing plants. Here, we performed an extensive analysis on the subcellular localization of NMTs from four distinct medicinal plants but also experimentally validated that two putative NMTs from Catharanthus roseus exhibit N-methyltransferase activity. Apart from providing unprecedented data regarding the targeting of these enzymes in planta, our results point out an additional layer of complexity to the subcellular organization of the MIA biosynthetic pathway by introducing tonoplast and peroxisome as new actors of the final steps of MIA biosynthesis.

Abstract 111: Conjoining Gene Expression And Cell Organization With Imaging To Explore Cell States In Hipsc-derived Cardiomyocytes

The Allen Institute for Cell Science is developing a state space of stem cell structural signatures to study changes in cellular organization of human induced pluripotent stem cells (hiPSCs) and other cell states through differentiation. Towards this goal, we have used CRISPR/Cas9 to generate a collection of ~50 endogenous fluorescently tagged hiPSC lines (www.allencell.org), each expressing a monoallelic EGFP-tagged protein that localizes to a particular cellular structure or organelle. In this study, we focuson hiPSC-derived cardiomyocytes and compare the relationship between sarcomeric structural organization and gene expression signatures at large scale. We developed several tools and novel quantitative approaches to achieve this: 1) scarless GFP-tagging of cardiac genes such as ACTN2 to study the organization and morphogenesis of the contractile apparatus; 2) a robust protocol for differentiation of hiPSCs into cardiomyocytes and methods for preparing cells for imaging; and 3) a quantitative, image-based platform for the systematic and automated classification of subcellular organization in single cells. We use these approaches to quantify subcellular organization and gene expression in >30,000 individual human induced pluripotent stem cell-derived cardiomyocytes, producing a publicly available dataset that describes the population distributions of local and global sarcomere organization, mRNA abundance, and correlations between these traits. While the mRNA abundance of some phenotypically important genes correlates with subcellular organization (e.g., MYH7), these two cellular metrics are heterogeneous and often uncorrelated, which suggests that geneexpression alone is not sufficient to classify cell states. Instead, we posit thatcell state should be defined by observing full distributions of quantitative, multidimensional traits in single cells that also account for space, time, and function. This platform provides a multidimensional approach to classify hiPSC-derived cardiomyocytes based on structural organization and gene expression in single cells.

The Phage Nucleus and PhuZ Spindle: Defining Features of the Subcellular Organization and Speciation of Nucleus-Forming Jumbo Phages

Bacteriophages and their bacterial hosts are ancient organisms that have been co-evolving for billions of years. Some jumbo phages, those with a genome size larger than 200 kilobases, have recently been discovered to establish complex subcellular organization during replication. Here, we review our current understanding of jumbo phages that form a nucleus-like structure, or “Phage Nucleus,” during replication. The phage nucleus is made of a proteinaceous shell that surrounds replicating phage DNA and imparts a unique subcellular organization that is temporally and spatially controlled within bacterial host cells by a phage-encoded tubulin (PhuZ)-based spindle. This subcellular architecture serves as a replication factory for jumbo Pseudomonas phages and provides a selective advantage when these replicate in some host strains. Throughout the lytic cycle, the phage nucleus compartmentalizes proteins according to function and protects the phage genome from host defense mechanisms. Early during infection, the PhuZ spindle positions the newly formed phage nucleus at midcell and, later in the infection cycle, the spindle rotates the nucleus while delivering capsids and distributing them uniformly on the nuclear surface, where they dock for DNA packaging. During the co-infection of two different nucleus-forming jumbo phages in a bacterial cell, the phage nucleus establishes Subcellular Genetic Isolation that limits the potential for viral genetic exchange by physically separating co-infection genomes, and the PhuZ spindle causes Virogenesis Incompatibility, whereby interacting components from two diverging phages negatively affect phage reproduction. Thus, the phage nucleus and PhuZ spindle are defining cell biological structures that serve roles in both the life cycle of nucleus-forming jumbo phages and phage speciation.

High resolution 3D imaging of liver reveals a central role for subcellular architectural organization in metabolism

Cells display complex intracellular organization through compartmentalization of metabolic processes into organelles, yet neither the resolution of these structures in the native tissue context nor its functional consequences are well understood. Here, we resolved the 3-dimensional organelle structural organization in large (>2.8×105μm3) volumes of intact liver tissue (15 partial or full hepatocytes per condition) in high resolution (8nm isotropic pixel size) by utilizing enhanced Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) imaging, followed by deep-learning-based image segmentation and 3D reconstruction. We also performed a comparative analysis of subcellular structures in liver tissue of lean and obese animals and found marked alterations particularly in hepatic endoplasmic reticulum (ER), which undergoes massive structural re-organization in obesity characterized by marked disorganization of stacks of ER sheets and predominance of ER tubules. Finally, we demonstrated the functional importance of these structural changes upon experimental recovery of the subcellular organization and its marked impact on cellular and systemic metabolism. We conclude that hepatic subcellular organization and ER’s architecture is highly dynamic, integrated with the metabolic state, and critical for adaptive homeostasis and tissue health.

Cell shape regulates subcellular organelle location to control early Ca2+ signal dynamics in vascular smooth muscle cells

The shape of the cell is connected to its function; however, we do not fully understand underlying mechanisms by which global shape regulates a cell’s functional capabilities. Using theory, experiments and simulation, we investigated how physiologically relevant cell shape changes affect subcellular organization, and consequently intracellular signaling, to control information flow needed for phenotypic function. Vascular smooth muscle cells going from a proliferative and motile circular shape to a contractile fusiform shape show changes in the location of the sarcoplasmic reticulum, inter-organelle distances, and differential distribution of receptors in the plasma membrane. These factors together lead to the modulation of signals transduced by the M3 muscarinic receptor/Gq/PLCβ pathway at the plasma membrane, amplifying Ca2+ dynamics in the cytoplasm, and the nucleus resulting in phenotypic changes, as determined by increased activity of myosin light chain kinase in the cytoplasm and enhanced nuclear localization of the transcription factor NFAT. Taken together, our observations show a systems level phenomenon whereby global cell shape affects subcellular organization to modulate signaling that enables phenotypic changes.

The emergence of phase separation as an organizing principle in bacteria

Recent investigations in bacteria suggest that membraneless organelles play a crucial role in the subcellular organization of bacterial cells. However, the biochemical functions and assembly mechanisms of these compartments have not yet been completely characterized. This Review assesses the current methodologies used in the study of membraneless organelles in bacteria, highlights the limitations in determining the phase of complexes in cells that are typically an order of magnitude smaller than a eukaryotic cell, and identifies gaps in our current knowledge about the functional role of membraneless organelles in bacteria. Liquid-liquid phase separation (LLPS) is one proposed mechanism for membraneless organelle assembly. Overall, we outline the framework to evaluate LLPS in vivo in bacteria, we describe the bacterial systems with proposed LLPS activity, and we comment on the general role LLPS plays in bacteria and how it may regulate cellular function. Lastly, we provide an outlook for super-resolution microscopy and single-molecule tracking as tools to assess condensates in bacteria.Statement of SignificanceThough membraneless organelles appear to play a crucial role in the subcellular organization and regulation of bacterial cells, the biochemical functions and assembly mechanisms of these compartments have not yet been completely characterized. Furthermore, liquid-liquid phase separation (LLPS) is one proposed mechanism for membraneless organelle assembly, but it is difficult to determine subcellular phases in tiny bacterial cells. Thus, we outline the framework to evaluate LLPS in vivo in bacteria and we describe the bacterial systems with proposed LLPS activity in the context of these criteria.