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.

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.

Clathrin adaptors mediate two sequential pathways of intra-Golgi recycling

The pathways of membrane traffic within the Golgi apparatus are not fully known. This question was addressed using the yeast Saccharomyces cerevisiae, in which the maturation of individual Golgi cisternae can be visualized. We recently proposed that the AP-1 clathrin adaptor mediates intra-Golgi recycling late in the process of cisternal maturation. Here, we demonstrate that AP-1 cooperates with the Ent5 clathrin adaptor to recycle a set of Golgi transmembrane proteins, including some that were previously thought to pass through endosomes. This recycling can be detected by removing AP-1 and Ent5, thereby diverting the AP-1/Ent5–dependent Golgi proteins into an alternative recycling loop that involves traffic to the plasma membrane followed by endocytosis. Unexpectedly, various AP-1/Ent5–dependent Golgi proteins show either intermediate or late kinetics of residence in maturing cisternae. We infer that the AP-1/Ent5 pair mediates two sequential intra-Golgi recycling pathways that define two classes of Golgi proteins. This insight can explain the polarized distribution of transmembrane proteins in the Golgi.

Membrane Traffic in Aspergillus oryzae and Related Filamentous Fungi

The industrially important filamentous fungus Aspergillus oryzae, known as the yellow Koji mold and also designated the Japanese National fungus, has been investigated for understanding the intracellular membrane trafficking machinery due to the great ability of valuable enzyme production. The underlying molecular mechanisms of the secretory pathway delineate the main secretion route from the hyphal tip via the vesicle cluster Spitzenkörper, but also there is a growing body of evidence that septum-directed and unconventional secretion occurs in A. oryzae hyphal cells. Moreover, not only the secretory pathway but also the endocytic pathway is crucial for protein secretion, especially having a role in apical endocytic recycling. As a hallmark of multicellular filamentous fungal cells, endocytic organelles early endosome and vacuole are quite dynamic: the former exhibits constant long-range motility through the hyphal cells and the latter displays pleiomorphic structures in each hyphal region. These characteristics are thought to have physiological roles, such as supporting protein secretion and transporting nutrients. This review summarizes molecular and physiological mechanisms of membrane traffic, i.e., secretory and endocytic pathways, in A. oryzae and related filamentous fungi and describes the further potential for industrial applications.

Promoters and Antagonists of Phagocytosis: A Plastic and Tunable Response

Recent observations indicate that, rather than being an all-or-none response, phagocytosis is finely tuned by a host of developmental and environmental factors. The expression of key phagocytic determinants is regulated via transcriptional and epigenetic means that confer memory on the process. Membrane traffic, the cytoskeleton, and inside-out signaling control the activation of phagocytic receptors and their ability to access their targets. An exquisite extra layer of complexity is introduced by the coexistence of distinct “eat-me” and “don't-eat-me” signals on targets and of corresponding “eat” and “don't-eat” receptors on the phagocyte surface. Moreover, assorted physical barriers constitute “don't-come-close-to-me” hurdles that obstruct the engagement of ligands by receptors. The expression, mobility, and accessibility of all these determinants can be modulated, conferring extreme plasticity on phagocytosis and providing attractive targets for therapeutic intervention in cancer, atherosclerosis, and dementia. Expected final online publication date for the Annual Review of Cell and Developmental Biology, Volume 37 is October 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Robustness and innovation along the endocytic route: Lessons from darkness

What mechanisms ensure the loading of a SNARE into a nascent carrier? In this issue, Bowman et al. (2021. J. Cell Biol.https://doi.org/10.1083/jcb.202005173) describe an unprecedented mechanism where two sorting complexes, AP-3 and BLOC-1, the latter bound to syntaxin 13, work as a fail-safe to recognize sorting signals in VAMP7, a membrane protein required for fusion to melanosomes. Their observations define one of the first examples of distributed robustness in membrane traffic mechanisms.

The acyltransferase lycat controls specific phosphoinositides and related membrane traffic and hormone receptor signaling

Phosphoinositdes (PIPs) are a group of signaling phospholipids involved in regulating many cellular processes, including organelle dynamics, nutrient uptake, autophagy and apoptosis. Through the action of lipid kinases and phosphatases, phosphatidylinositol (PI) can be phosphorylated on three different positions of the inositol headgroup resulting in seven distinct PIP species. Substantial research has focused on elucidating the function and importance of headgroup phosphorylation while much less is known about the significance of the incorporation of specific acyl chains within PI. PI exhibits unique specificity of acyl chain composition, where majority contains 1-stearoyl-2-arachidonoyl acyl species. This unique acyl chain enrichment is, in part, controlled by the PI acyltransferase lysocardiolipin acyltransferase (LYCAT). How LYCAT and, in turn, incorporation of specific fatty acids, controls the function of PI and PIPs is poorly understood. Thus, I investigated the impact of LYCAT perturbation on PIP acyl profile and effects on PIP-dependent processes. Perturbation of LYCAT by siRNA gene silencing resulted in a shift in the acyl profile of PIP2 species to contain shorter species. Additionally, LYCAT silencing altered the cellular localization and levels of phosphatidylinositol-4,5-bisphosphate and phosphatidylinositol-3-phosphate but was without effect on other PI species examined. Consistent with this, silencing of LYCAT perturbed the membrane traffic of transferrin receptor dependent on these specific PIPs. I also observed changes in PI-dependent receptor tyrosine kinase signaling pathways that control cell survival and proliferation, which are regulated by phosphatidylinositol-3,4,5-trisphosphate. LYCAT perturbation altered activation of Akt1, which impacted a number of Akt substrates. Additionally, using fluorescence microscopy, I discovered that LYCAT is localized to peripheral ER vesicles that contain PI synthase enzyme, which is responsible for PI synthesis. These peripheral vesicles partially overlap with endoplasmic reticulum-plasma membrane contact sites marked by E-Syt2 but showed little overlap with the ER maker, KDEL. Collectively, my results show that the PI acyltransferase LYCAT controls the function of specific species of PIPs, which in turn selectively impacts specific stages of endomembrane traffic and hormone receptor signaling. Hence, the regulation of acyl content of PI is an important new dimension for the control of PI and PIP function.

Control Of Phosphoinositide Synthesis And Degradation By The Acyltransferase Lycat

Phosphoinositides (PIPs) are important regulators of various cellular phenomena including intracellular signaling, membrane traffic and cell migration. PIPs are formed as a result of the regulated phosphorylation of the inositol headgroup of phosphatidylinositol (PI) on specific positions by certain lipid kinases and phosphatases. It is well appreciated that the enrichment of specific PIPs, defined by inositol headgroup phosphorylation, within specific membrane compartments plays a critical role in organelle identity and membrane traffic. However, while much attention has been given to understanding of the role of inositol headgroup phosphorylation in PIP function, much less is known about the role of dynamic incorporation of specific acyl groups into these phospholipids. Importantly, PI and PIPs exhibit remarkable and unique selectivity for certain acyl groups. For example, about 45% of PIs (but not other phospholipids) are rich in 1-steroyl 2-arachidonyl. We recently identified that the possible control of the selective incorporation of steric acid at the sn-1 position is by the acyltransferase LYCAT, which controls the levels, acyl profile and function of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) (Bone et al. Mol Biol Cell 2017. 28:161-172). Here we examine how perturbation of LYCAT leads to a reduction in the levels of PI(4,5)P2 and phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P3). To measure the rate of PI(4,5)P2 synthesis, we treated cells with ionomycin to first ablate this PIP, followed by washout of the drug and monitoring of rate of reappearance via localization of a fluorescent PI(4,5)P2 probe. To measure the rate of PI(4,5)P2 degradation, we arrested PI(4,5)P2 synthesis by a pharmacological inhibitor, phenylarsine oxide (PAO) and monitored the loss of cellular PI(4,5)P2. Lastly, to examine the production of PI(3,4,5)P3, we treated cells with epidermal growth factor (EGF) and monitored the production of this PIP. Together, this work provides new information about how the dynamic and selective remodeling of specific phospholipids controls their levels, localization and function.