Human Cytomegalovirus Hijacks WD Repeat Domain 11 for Virion Assembly Compartment Formation and Virion Morphogenesis

Human cytomegalovirus (HCMV) has a large (∼235-kb) genome with over 200 predicted open reading frames and exploits numerous cellular factors to facilitate its replication. A key feature of HCMV-infected cells is the emergence of a distinctive membranous cytoplasmic compartment termed the virion assembly compartment (vAC). Here we report that host protein WD repeat domain 11 (WDR11) plays a key role in vAC formation and virion morphogenesis. We found that WDR11 was up-regulated at both mRNA and protein levels during HCMV infection. At the late stage of HCMV replication, WDR11 relocated to the vAC and co-localized with markers of the trans-Golgi network (TGN) and vAC. Depletion of WDR11 hindered HCMV-induced membrane reorganization of the Golgi and TGN, altered vAC formation, and impaired HCMV secondary envelopment and virion morphogenesis. Further, motifs critical for the localization of WDR11 in TGN were identified by alanine-scanning mutagenesis. Mutation of these motifs led to WDR11 mislocation outside of the TGN and loss of vAC formation. Taken together, these data indicate that host protein WDR11 is required for efficient viral replication at the stage of virion assembly, possibly by facilitating the remodeling of the endomembrane system for vAC formation and virion morphogenesis. Importance During the late phase of human cytomegalovirus (HCMV) infection, the endomembrane system is dramatically reorganized, resulting in the formation of a unique structure termed the virion assembly compartment (vAC), which is critical for the assembly of infectious virions. The mechanism of HCMV-induced vAC formation is still not fully understood. In this report, we identified a host factor, WDR11, that plays an important role in vAC formation. Our findings argue that WDR11 contributes to the relocation of the Golgi and trans-Golgi network to the vAC, a membrane reorganization process that appears to be required for efficient virion maturation. The present work provides new insights into the vAC formation and HCMV virion morphogenesis and a potential novel target for anti-viral treatment.

Proteomics of isolated sieve tubes from Nicotiana tabacum: sieve element–specific proteins reveal differentiation of the endomembrane system

Symplasmicly connected cells called sieve elements form a network of tubes in the phloem of vascular plants. Sieve elements have essential functions as they provide routes for photoassimilate distribution, the exchange of developmental signals, and the coordination of defense responses. Nonetheless, they are the least understood main type of plant cells. They are extremely sensitive, possess a reduced endomembrane system without Golgi apparatus, and lack nuclei and translation machineries, so that transcriptomics and similar techniques cannot be applied. Moreover, the analysis of phloem exudates as a proxy for sieve element composition is marred by methodological problems. We developed a simple protocol for the isolation of sieve elements from leaves and stems of Nicotiana tabacum at sufficient amounts for large-scale proteome analysis. By quantifying the enrichment of individual proteins in purified sieve element relative to bulk phloem preparations, proteins of increased likelyhood to function specifically in sieve elements were identified. To evaluate the validity of this approach, yellow fluorescent protein constructs of genes encoding three of the candidate proteins were expressed in plants. Tagged proteins occurred exclusively in sieve elements. Two of them, a putative cytochrome b561/ferric reductase and a reticulon-like protein, appeared restricted to segments of the endoplasmic reticulum (ER) that were inaccessible to green fluorescent protein dissolved in the ER lumen, suggesting a previously unknown differentiation of the endomembrane system in sieve elements. Evidently, our list of promising candidate proteins (SI Appendix, Table S1) provides a valuable exploratory tool for sieve element biology.

Cell3: a new vision for study of the endomembrane system in mammalian cells

The endomembrane system of mammalian cells provides massive capacity for the segregation of biochemical reactions into discrete locations. The individual organelles of the endomembrane system also require the ability to precisely transport material between these compartments in order to maintain cell homeostasis; this process is termed membrane traffic. For several decades, researchers have been systematically identifying and dissecting the molecular machinery that governs membrane trafficking pathways, with the overwhelming majority of these studies being carried out in cultured cells growing as monolayers. In recent years, a number of methodological innovations have provided the opportunity for cultured cells to be grown as 3-dimensional (3D) assemblies, for example as spheroids and organoids. These structures have the potential to better replicate the cellular environment found in tissues and present an exciting new opportunity for the study of cell function. In this mini-review, we summarize the main methods used to generate 3D cell models and highlight emerging studies that have started to use these models to study basic cellular processes. We also describe a number of pieces of work that potentially provide the basis for adaptation for deeper study of how membrane traffic is coordinated in multicellular assemblies. Finally, we comment on some of the technological challenges that still need to be overcome if 3D cell biology is to become a mainstream tool toward deepening our understanding of the endomembrane system in mammalian cells.

Un cómic ilustrativo sobre el tránsito intracelular de los virus.

El mecanismo de cómo los virus atraviesan las estructuras intracelulares son el modelo que ejemplifica el tránsito intracelular por el sistema de endomembranas, el mismo que es utilizado en la descripción de los diferentes aparatos intracelulares. El objetivo fue desarrollar un método didáctico que explique un mecanismo de la biología molecular, como lo es la tira cómica en el tránsito y la señalización intracelular de los virus. Se diseñó una historia cómica-ilustrativa donde una estructura viral es personificada por un espía encubierto, el eje fundamental de la trama es la obtención de las moléculas de información genética a nivel del núcleo, para ello tiene que pasar por los varios departamentos de la célula que representan las organelas intracitoplasmáticas con sus características funcionales, la secuencia obedece a la comunicación que tienen las diferentes organelas desde la membrana celular hasta el núcleo. La elaboración de comics o historietas pueden recrear eventos y fenómenos estudiados en las ciencias médicas como la biología, teniendo los siguientes resultados: aumentar el interés, la comprensión, la creatividad, la necesidad de conocimientos y generación de nuevas ideas en los estudiantes. El cómic es una herramienta didáctica útil en la enseñanza del tránsito intracelular a partir de la narración gráfica de un modelo viral que se transporta al interior de la célula. The mechanism of how viruses cross intracellular structures is the model that exemplifies intracellular transit through the endomembrane system, the same that is used in the description of the different intracellular apparatus. The objective was to develop a didactic method that explains a mechanism of molecular biology, such as the comic strip in the transit and intracellular signaling of viruses. A comic-illustrative story was designed where a viral structure is personified by an undercover spy, the fundamental axis of the plot is obtaining the genetic information molecules at the nucleus level, for this it has to go through the various departments of the cell that represent the intracytoplasmic organelles with their functional characteristics, the sequence obeys the communication that the different organelles have from the cell membrane to the nucleus. The development of comics or comics can recreate events and phenomena studied in medical sciences such as biology, having the following results: increase interest, understanding, creativity, the need for knowledge and generation of new ideas in students. The comic is a useful didactic tool in teaching intracellular transit from the graphic narration of a viral model that is transported inside the cell.

Folding and Insertion of Transmembrane Helices at the ER

In eukaryotic cells, the endoplasmic reticulum (ER) is the entry point for newly synthesized proteins that are subsequently distributed to organelles of the endomembrane system. Some of these proteins are completely translocated into the lumen of the ER while others integrate stretches of amino acids into the greasy 30 Å wide interior of the ER membrane bilayer. It is generally accepted that to exist in this non-aqueous environment the majority of membrane integrated amino acids are primarily non-polar/hydrophobic and adopt an α-helical conformation. These stretches are typically around 20 amino acids long and are known as transmembrane (TM) helices. In this review, we will consider how transmembrane helices achieve membrane integration. We will address questions such as: Where do the stretches of amino acids fold into a helical conformation? What is/are the route/routes that these stretches take from synthesis at the ribosome to integration through the ER translocon? How do these stretches ‘know’ to integrate and in which orientation? How do marginally hydrophobic stretches of amino acids integrate and survive as transmembrane helices?

Cell3: A new vision for study of the endomembrane system in mammalian cells

The endomembrane system of mammalian cells provides massive capacity for the segregation of biochemical reactions into discrete locations. The individual organelles of the endomembrane system also require the ability to precisely transport material between these compartments in order to maintain cell homeostasis; this process is termed membrane traffic. For several decades, researchers have been systematically identifying and dissecting the molecular machinery that governs membrane trafficking pathways, with the overwhelming majority of these studies being carried out in cultured cells growing as monolayers. In recent years, a number of methodological innovations have provided the opportunity for cultured cells to be grown as 3-dimensional (3D) assemblies, for example as spheroids and organoids. These structures have the potential to better replicate the cellular environment found in tissues, and present an exciting new opportunity for the study of cell function. In this mini-review we summarise the main methods used to generate 3D cell models, and highlight emerging studies that have started to use these models to study basic cellular processes. We also describe a number of pieces of work that potentially provide the basis for adaptation for deeper study of how membrane traffic is coordinated in multicellular assemblies. Finally, we comment on some of the technological challenges that still need to be overcome if 3D cell biology is to become a mainstream tool towards deepening our understanding of the endomembrane system in mammalian cells.

The role of clathrin in exocytosis and the mutual regulation of endo- and exocytosis in plant cells

Within the plant endomembrane system, the vesicle coat protein clathrin localizes to the plasma membrane (PM) and the trans-Golgi Network/Early Endosome (TGN/EE). While the role of clathrin as a major component of endocytosis at the PM is well established, its function at TGN/EE, possibly in exocytosis or the vacuolar pathway, is a matter of debate. This shared function of clathrin also opens a question whether plant cells possess a homeostatic mechanisms that balance rates of opposite trafficking routes, such as endo- and exocytosis. Here we address these questions using lines inducibly silencing CLATHRIN HEAVY CHAIN (CHC). We find a relocation of exocytic soluble and integral membrane protein cargoes to the vacuole, supporting a function of clathrin in exocytosis. A comparison with lines overexpressing AUXILIN-LIKE1, where inhibition of CME precedes rerouting of secretory cargoes, does not support a homeostatic regulatory mechanism adjusting exocytosis to the rates of endocytosis. Complementary experiments reveal only minor and variably detectable reductions in the rates of CME in secretory mutants, also not indicative of a converse homeostatic mechanism adjusting rates of endocytosis to the rates of secretion.

Mitochondria transplantation between living cells

Mitochondria and the complex endomembrane system are hallmarks of eukaryotic cells. To date, it has been difficult to manipulate organelle structures within single live cells. We developed a FluidFM-based approach to extract, inject and transplant organelles from and into living cells with subcellular spatial resolution. The approach enabled the transfer of controlled quantities of mitochondria into cells while maintaining their viability and monitoring their fate in new host cells. Transplantation of healthy and drug-impaired mitochondria into primary keratinocytes allowed real-time tracking of mitochondrial subpopulation rescue. Fusion with the mitochondrial network of recipient cells occurred 20 min after transplantation and continued for over 16 hours. After transfer of mitochondria and cell propagation over generations, we show that donor mtDNA was replicated in recipient cells without the need for selection pressure. The approach opens new prospects for the study of organelle physiology and homeostasis, but also for mechanobiology, synthetic biology, and therapy.

A propósito del tránsito intracelular de los virus. Comic ilustrativo.

Introduction. The mechanism of how viruses cross intracellular structures is the model that exemplifies the intracellular transit through the endomembrane system, the same that is used in the teaching and description of the different intracellular apparatus and organelles. Objective. Develop a didactic method that explains a mechanism of molecular biology, such as the comic strip in the transit and intracellular signaling of viruses. Methodology. A comic-illustrative story was designed where a viral structure is personified by an undercover spy, the fundamental axis of the plot is obtaining the genetic information molecules at the nucleus level, for this it has to go through the various departments of the cell representing the intracellular and intracytoplasmic organelles with their functional characteristics, the sequence obeys the communication that the different organelles have from the cell membrane to the nucleus. Results. The development of comics or comics can recreate events and phenomena studied in medical sciences such as biology, having the following results: increase interest, understanding, creativity, the need for knowledge and generation of new ideas in students. Conclusion. The comic is a useful didactic tool in teaching intracellular transit from the graphic narration of a viral model that is transported inside the cell.

LRRK2 along the Golgi and lysosome connection: a jamming situation

Parkinson's disease (PD) is an age-related neurodegenerative disorder, clinically characterized by bradykinesia, rigidity, and resting tremor. Leucine-Rich Repeat Kinase 2 (LRRK2) is a large, multidomain protein containing two enzymatic domains. Missense mutations in its coding sequence are amongst the most common causes of familial PD. The physiological and pathological impact of LRRK2 is still obscure, but accumulating evidence supports a role for LRRK2 in membrane and vesicle trafficking, mainly functioning in the endosome-recycling system, (synaptic) vesicle trafficking, autophagy, and lysosome biology. LRRK2 binds and phosphorylates key regulators of the endomembrane systems and is dynamically localized at the Golgi. The impact of LRRK2 on the Golgi may reverberate throughout the entire endomembrane system and occur in multiple intersecting pathways, including endocytosis, autophagy, and lysosomal function. This would lead to overall dysregulation of cellular homeostasis and protein catabolism, leading to neuronal dysfunction and accumulation of toxic protein species, thus underlying the possible neurotoxic effect of LRRK2 mutations causing PD.