ER membranes exhibit phase behavior at sites of organelle contact

Abstract

The plasma membrane of cells exhibits phase behavior that allows transient concentration of specific proteins and lipids, giving rise to functionally dynamic and diverse nanoscopic domains. This phase behavior is observable in giant plasma membrane-derived vesicles, in which microscopically visible, liquid-ordered (Lo) and liquid-disordered (Ld) lipid domains form upon a shift to low temperatures. The extent such phase behavior exists in the membrane of the endoplasmic reticulum (ER) of cells remains unclear. To explore the phase behavior of the ER membrane in cells, we used hypotonic cell swelling to generate Large Intra-Cellular Vesicles (LICVs) from the ER in cells. ER LICVs retained their lumenal protein content, could be retubulated into an ER network, and maintained stable inter-organelle contacts, where protein tethers are concentrated at these contacts. Notably, upon temperature reduction, ER LICVs underwent reversible phase separation into microscopically-visible Lo and Ld lipid domains. The Lo lipid domains marked ER contact sites with other organelles. These findings demonstrate thatLICVs provide an important model system for studying the biophysical properties of intracellular organelles in cells. Significance StatementPrior work has demonstrated that the plasma membrane can phase separate into microscopically visible Lo and Ld domains with distinct lipid and protein content. However, such behavior on the ER membrane has not been experimentally observed, even though the ER contacts every organelle of the cell, exchanging lipids and metabolites in a highly regulated manner at these contacts. We find here that hypotonic treatment generates Large Intra-Cellular Vesicles from the endoplasmic reticulum and other membrane-bound organelles in cells, enabling the study of phase behavior on the ER membrane. We show that ER membranes can be reversibly phase separated into microscopically-observable, Lo and Ld domains. ER LICVs also maintained stable inter-organelle contact sites in cells, with organelle tethers concentrated at these contacts.

Self‐assembly of multi‐component mitochondrial nucleoids via phase separation

“…Our results suggest that the interaction of mt-nucleoids with membranes is due to the emergent wetting behavior of the condensate on the membrane (Snead & Gladfelter, 2019). Similar observations have been made for contact sites between tethering proteins from various membrane bound organelles, including the mitochondrial mitofusin 1 (Mfn1) tethering protein and Sec61β of the ER membrane (King, Sengupta et al, 2020), and for the condensation of Atg1-complex droplets, as part of the pre-autophagosomal structure (PAS), along vacuolar membranes (Fujioka, Alam et al, 2020). It is tempting to speculate that the wetting behavior between the mitochondrial inner membrane and the mt-nucleoid may not only play a role in regulating the size and diffusion of mt-nucleoids, but also in functional processes such as replication (Lewis, Uchiyama et al, 2016).…”

Axonal Organelles as Molecular Platforms for Axon Growth and Regeneration after Injury

“…The ER is composed of two distinct interconnected structures-ER tubules and flattened ER sheets. The latter could, in some cases represent a densely packaged network of tubules [24,25,26]. This structure supports a highly dynamic model where the ER can rapidly change its structure and distribution to meet any changing cellular demands and to modulate its interactions with various other organelles throughout the cell, making it a perfect candidate for a role in axon growth and regeneration.…”

“…The proper localization and functioning of mitochondria are important to meet the high energy demands of developing neurons and to support neuronal processes such as axon growth and neurotransmission. Recent advances in imaging have allowed for the visualization of mitochondria in vitro and in intact organisms such as mice and zebrafish [26,31,107,108,109,110,111]. Similar to other organelles, mitochondrial transport into the axon is mostly microtubule-based and relies on motor proteins such as kinesin and dynein as well as mitochondria-specific adaptor proteins such as Milton and Miro [112,113,114,115,116].…”

“…Inter-organellar interactions have been historically difficult to study due to their dynamic nature and due to their low temporal and spatial resolution. Only recently, with the advancement of high-resolution microscopy, could the complexity of this interconnectivity be studied [25,26]. In fibroblasts, multi-level interactions between six different organelles were recently observed by confocal and lattice light sheet microscopy [252].…”

The evolution of a cell biologist

“…My lab has branched out into several new areas ( Figure 2 ). We are studying sites of mRNA translation, viral budding, interorganelle contacts, phase condensate dynamics, and cell–cell fusion, while maintaining an interest in classic organelles like ER, Golgi, mitochondria, and lysosomes ( Nixon-Abell et al , 2016 ; Seo et al , 2017 ; Valm et al , 2017 ; Cai et al , 2019 ; Chang et al , 2019 ; King et al , 2019 ; Liao et al , 2019 ; Sengupta et al , 2019 ). Janelia’s unique combination of four-dimensional imaging capabilities, chemical probe development, protein-based sensor engineering, and innovative microscopes has proven to be an ideal environment.…”