Membrane Reservoir Research Articles

Time-resolved proximity proteomics uncovers a membrane tension-sensitive caveolin-1 interactome at the rear of migrating cells.

Caveolae are small membrane pits with fundamental roles in mechanotransduction. Several studies have shown that caveolae flatten out in response to an increase in membrane tension, thereby acting as a mechanosensitive membrane reservoir that buffers acute mechanical stress. The dynamic assembly and disassembly of caveolae has also been implicated in the control of RhoA/ROCK-mediated actomyosin contractility at the rear of migrating cells. However, how membrane tension controls the organisation of caveolae and caveolae-mediated mechanotransduction is poorly understood. To address this, we systematically quantified protein-protein interactions of caveolin-1 in migrating RPE1 cells at steady state and in response to an acute increase in membrane tension using biotin-based proximity labelling and quantitative mass spectrometry. Our data show that caveolae are highly enriched at the rear of migrating RPE1 cells and that membrane tension rapidly and reversibly disassembles the caveolar protein coat. Membrane tension also dislodges caveolin-1 from focal adhesion proteins and several mechanosensitive cortical actin regulators including filamins and cortactin. In addition, we present evidence that ROCK and the RhoGAP ARHGAP29 are associated with caveolin-1 in a membrane tension-dependent manner, and that ARHGAP29 regulates caveolin-1 Y14 phosphorylation, caveolae rear localisation, and RPE1 cell migration. Taken together, our work uncovers a membrane tension-sensitive coupling between caveolae and the rear-localised F-actin cytoskeleton. This provides a framework for dissecting the molecular mechanisms underlying caveolae-regulated mechanotransduction pathways.

Linear podosomes display low Cdc42 activity for proplatelet elongation by megakaryocytes

Blood platelets result from differentiation of megakaryocytes (MKs) into the bone marrow. It culminates with the extension of proplatelets (PPT) through medullar sinusoids and release of platelets in the blood stream. Those processes are regulated by contact with the microenvironment mediated by podosomes. We previously demonstrated that contact of megakaryocytes to Collagen I fibers initiated the formation of linear podosomes required for proplatelets extension and release of mature platelets. MKs linear podosomes have the particularity of displaying mechanical pulling activity but, unlike other linear podosomes, they lack the ability of digesting the extracellular matrix (ECM), as we recently demonstrated. The Cdc42 small GTPase is required for actomyosin-dependent maturation of the demarcation membrane system (DMS), a membrane reservoir for PPT and platelets components. Cdc42 is a known protein of the podosomes core, and is instrumental to accurate platelets release into the sinusoids. Indeed, FRET analysis showed that Cdc42 activity was very high and central to DMS formation. Unexpectedly, even though we found the protein in linear podosomes, almost undetectable Cdc42 activity was detected in those structures. This observation suggests that Cdc42 could also act as scaffold to assemble proteins required for PPT formation/elongation along Collagen I fibers in MKs. Eventually, we demonstrated that linear podosomes appear as points of contact between Collagen I fibers and DMS membranes, to mechanically extend PPT along Collagen bundles, independently of Cdc42 activity.

Developing a synergistic rate-retarding polymeric implant for controlling monoclonal antibody delivery in minimally invasive glaucoma surgery

Monoclonal antibodies (mAbs) have garnered substantial attention within the field of ophthalmology and can be used to suppress scar formation after minimally invasive glaucoma surgeries. Here, by controlling mAb passive diffusion, we developed a polymeric, rate-controlling membrane reservoir loaded with poly(lactic-co-glycolic acid) microspheres to deliver mAb for several weeks. Different parameters were tested to ensure that the microspheres achieved a good quality characteristic, and our results showed that 1 %W/V emulsifier with 5 %W/V NaCl achieved mAb-loaded microspheres with the highest stability, encapsulation efficiency and minimal burst release. Then, we fabricated and compared 10 types of microporous films based on polylactic acid (PLA), polycaprolactone (PCL), and polyethylene glycol (PEG). Our results revealed distinct pore characteristics and degradation patterns in different films due to varying polymer properties, and all the polymeric film formulations showed good biocompatibility in both human trabecular meshwork cells and human conjunctival fibroblasts. Finally, the optimized microspheres were loaded into the reservoir-type polymeric implant assembled by microporous membranes with different surface coating modifications. The implant formulation, which was fabricated by 60 PCL: 40 PEG (3 %W/V) polymer with 0.1 %W/V poly(lactic-co-glycolic acid) barrier, exerted the best drug release profile that can sustained release mAb (83.6 %) for 4 weeks.

Salmonella exploits membrane reservoirs for invasion of host cells

Salmonella utilizes a type 3 secretion system to translocate virulence proteins (effectors) into host cells during infection1. The effectors modulate host cell machinery to drive uptake of the bacteria into vacuoles, where they can establish an intracellular replicative niche. A remarkable feature of Salmonella invasion is the formation of actin-rich protuberances (ruffles) on the host cell surface that contribute to bacterial uptake. However, the membrane source for ruffle formation and how these bacteria regulate membrane mobilization within host cells remains unclear. Here, we show that Salmonella exploits membrane reservoirs for the generation of invasion ruffles. The reservoirs are pre-existing tubular compartments associated with the plasma membrane (PM) and are formed through the activity of RAB10 GTPase. Under normal growth conditions, membrane reservoirs contribute to PM homeostasis and are preloaded with the exocyst subunit EXOC2. During Salmonella invasion, the bacterial effectors SipC, SopE2, and SopB recruit exocyst subunits from membrane reservoirs and other cellular compartments, thereby allowing exocyst complex assembly and membrane delivery required for bacterial uptake. Our findings reveal an important role for RAB10 in the establishment of membrane reservoirs and the mechanisms by which Salmonella can exploit these compartments during host cell invasion.

Subconductance states in a semimicroscopic model for a tetrameric pore.

A physical model for a structured tetrameric pore is studied. The pore is modeled as a device composed of four subunits, each one exhibiting two possible states (open and closed). The pore is located within a membrane that separates two reservoirs with ionic solutions. All variables of the model follow physical dynamical equationsaccounting for the internal structure of the pore, derived from a single energy functional and supplemented with thermal noises. An extensive study of the resulting ionic intensity is performed for different values of the control parameters, mainly membrane potential and reservoir ion concentrations. Two possible physical devices are studied: voltage-gated (including a voltage sensor in each subunit) and non-voltage-gated pores. The ionic flux through the pore exhibits several distinct dynamical configurations, in particular subconductance states, which indicate very different dynamical internal states of the subunits. Such subconductance states become much easier to observe in sensorless pores. These results are compared with available experimental data on tetrameric K channels and analytical predictions.

PACSIN2 Orchestrates Actin Reorganization during Megakaryocyte Maturation

Thrombocytopenia, defined by a platelet count below 150,000/µL, poses significant mortality risks, primarily due to bleeding. Blood platelets are produced by bone marrow megakaryocytes (MKs) in unique cellular processes that require polyploidization and extensive intracellular membrane rearrangements leading to the formation of the demarcation membrane system (DMS), the surface-connected membrane reservoir for future platelets. The membrane-deforming F-BAR protein PACSIN2 is one of the most abundant and conserved BAR proteins in platelets, and exomechip analysis and genome-wide association studies have associated PACSIN2 single nucleotide variants with platelet count and mean platelet volume in humans. Our previous work has shown that PACSIN2 localizes with the initiating DMS in MKs in a process regulated by the cytoskeletal and scaffolding protein filamin A (FlnA). Recently, we have found that Pacsin2 -/- mice display mild thrombocytopenia with slightly enlarged platelets and severe thrombus formation defects due to increased activation of platelet integrin β1. Here we investigated the role of PACSIN2 in MK maturation and platelet production. Cultured fetal liver cell-derived Pacsin2 -/- MKs formed proplatelets normally compared to controls. Pacsin2 -/- bone marrow sections displayed MK count comparable to control mice, and freshly isolated bone marrow Pacsin2 -/- MKs had normal ploidy, indicating normal MK maturation. The observed effects of PACSIN2 deficiency led to a defective formation of the DMS within MKs. In PACSIN2 deficient MKs, the DMS exhibited a less distinct structure, making it challenging to delineate clear platelet territories. Interestingly, the lack of PACSIN2 also impacted the spatial distribution of GPIbα and integrin β1 membrane receptors. These receptors, typically dispersed within the cytoplasm in normal MKs, were concentrated at the cell periphery in Pacsin2 -/-MKs. A similar relocation was observed for polymerized actin. This finding underscores the pivotal role of PACSIN2 in actin reorganization within MKs, which in turn influences the rearrangement of GPIbα and integrin β1. The data imply that receptor and actin cytoskeleton rearrangements are critical to DMS formation and subsequent platelet production in vivo.

A palisade-shaped membrane reservoir is required for rapid ring cell inflation in Drechslerella dactyloides

Fusion of individual vesicles carrying membrane-building materials with the plasma membrane (PM) enables gradual cell expansion and shape change. Constricting ring (CR) cells of carnivorous fungi triple in size within 0.1-1 s to capture passing nematodes. Here, we investigated how a carnivorous fungus, Drechslerella dactyloides, executes rapid and irreversible PM expansion during CR inflation. During CR maturation, vesicles carrying membrane-building materials accumulate and fuse, forming a structure named the Palisade-shaped Membrane-building Structure (PMS) around the rumen side of ring cells. After CR inflation, the PMS disappears, with partially inflated cells displaying wavy PM and fully inflated cells exhibiting smooth PM, suggesting that the PMS serves as the reservoir for membrane-building materials to enable rapid and extensive PM expansion. The DdSnc1, a v-SNARE protein, accumulates at the inner side of ring cells and is necessary for PMS formation and CR inflation. This study elucidates the unique cellular mechanisms underpinning rapid CR inflation.

Cell migration in dense microenvironments.

The nucleus has been viewed as a passenger during cell migration that functions merely to protect the genome. However, increasing evidence shows that the nucleus is an active organelle, constantly sensing the surrounding environment and translating extracellular mechanical inputs into intracellular signaling. The nuclear envelope has a large membrane reservoir which serves as a buffer for mechanical inputs as it unfolds without increasing its tension. In contrast, when cells cope with mechanical strain, such as migration through solid tumors or dense interstitial spaces, the nuclear envelope folds stretch, increasing nuclear envelope tension and sometimes causing rupture. Different degrees of nuclear envelope tension regulate cellular behaviors and functions, especially in cells that move and grow within dense matrices. The crosstalk between extracellular mechanical inputs and the cell nucleus is a critical component in the modulation of cell function of cells that navigate within packed microenvironments. Moreover, there is a link between regimes of nuclear envelope unfolding and different cellular behaviors, from orchestrated signaling cascades to cellular perturbations and damage.

Consumption of a polarized membrane reservoir drives asymmetric membrane expansion during the unequal divisions of neural stem cells.

The asymmetric divisions of Drosophila neural stem cells (NSCs) produce unequally sized siblings, with most volume directed into the sibling that retains the NSC fate. Sibling size asymmetry results from the preferential expansion of the NSC sibling surface during division. Here, we show that a polarized membrane reservoir constructed by the NSC in early mitosis provides the source for expansion. The reservoir is formed from membrane domains that contain folds and microvilli that become polarized by apically directed cortical flows of actomyosin early in mitosis. When furrow ingression begins and internal pressure increases, the stores of membrane within the apical reservoir are rapidly consumed. Expansion is substantially diminished in NSCs that lack a reservoir, and membrane expansion equalizes when the reservoir is not polarized. Our results suggest that the cortical flows that remodel the plasma membrane during asymmetric cell division function to satisfy the dynamic surface area requirements of unequally dividing cells.

A dynamic mechanically stabilized membrane reservoir mediates fast endocytosis.

We present experimental, data analytic and mathematical modeling results suggesting repeated exocytic stimulation mechanically stabilizes an internal reservoir of plasma-membrane-attached vesicles serving as a reservoir for fast endocytic internalization.

Caveolin-1β promotes the production of active human microsomal glutathione S-transferase in induced intracellular vesicles inSpodoptera frugiperda21 insect cells

The heterologous expression in Spodoptera frugiperda 21 (Sf21) insect cells of the β isoform of canine caveolin-1 (caveolin-1β), using a baculovirus-based vector, resulted in intracellular vesicles enriched in caveolin-1β. We investigated whether these vesicles could act as membrane reservoirs, and promote the production of an active membrane protein (MP) when co-expressed with caveolin-1β. We chose hMGST1 (human microsomal glutathione S-transferase 1) as the co-expressed MP. It belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family of integral MPs, and, as a phase II detoxification enzyme, it catalyzes glutathione conjugation of lipophilic drugs present in the lipid membranes. In addition to its pharmaceutical interest, its GST activity can be conveniently measured. The expression of both MPs were followed by Western blots and membrane fractionation on density gradient, and their cell localization by immunolabeling and transmission electron microscopy. We showed that caveolin-1β kept its capacity to induce intracellular vesicles in the host when co-expressed with hMGST1, and that hMGST1 is in part addressed to these vesicles. Remarkably, a fourfold increase in the amount of active hMGST1 was found in the most enriched membrane fraction, along with an increase of its specific activity by 60% when it was co-expressed with caveolin-1β. Thus, heterologously expressed caveolin-1β was able to induce cytoplasmic vesicles in which a co-expressed exogenous MP is diverted and sequestered, providing a favorable environment for this cargo.

Tracheal tube fusion in Drosophila involves release of extracellular vesicles from multivesicular bodies

Extracellular vesicles (EVs) comprise diverse types of cell-released membranous structures that are thought to play important roles in intercellular communication. While the formation and functions of EVs have been investigated extensively in cultured cells, studies of EVs in vivo have remained scarce. We report here that EVs are present in the developing lumen of tracheal tubes in Drosophila embryos. We define two distinct EV subpopulations, one of which contains the Munc13-4 (also known as UNC13D) homolog Staccato (Stac) and is spatially and temporally associated with tracheal tube fusion (anastomosis) events. The formation of Stac-positive luminal EVs depends on the tracheal tip-cell-specific GTPase Arl3 (also known as Dnd in Drosophila), which is also required for the formation of Stac-positive multivesicular bodies (MVBs), suggesting that Stac-positive EVs derive from fusion of Stac-positive MVBs with the luminal membrane in tip cells during anastomosis formation. The GTPases Rab27 and Rab35 cooperate downstream of Arl3 to promote Stac-positive MVB formation and tube fusion. We propose that Stac-positive MVBs act as membrane reservoirs that facilitate tracheal lumen fusion in a process regulated by Arl3, Rab27, Rab35 and Stac. This article has an associated First Person interview with the first author of the paper.

Amniotic fluid surfactant: a new approach to the study of structure and function of pulmonary surfactant

To allow breathing dynamics, the lung requires a substance able to overwhelm the surface tension of alveoli. This substance, called pulmonary surfactant, is synthetized as tightly packed multi-layered lipid assemblies that have optimal adsorption capabilities to the air-liquid interface. Pulmonary surfactant forms a surface-active monolayer lining the respiratory surface, connected to a membrane reservoir that nurtures the interface with new lipids when it is needed. The two hydrophobic pulmonary surfactant proteins, SP-B and SP-C, optimize the structure of surfactant complexes and enhance its surface-active properties.

PACSIN proteins in vivo: Roles in development and physiology.

Protein kinase C and casein kinase substrate in neurons (PACSINs), or syndapins (synaptic dynamin‐associated proteins), are a family of proteins involved in the regulation of cell cytoskeleton, intracellular trafficking and signalling. Over the last twenty years, PACSINs have been mostly studied in the in vitro and ex vivo settings, and only in the last decade reports on their function in vivo have emerged. We first summarize the identification, structure and cellular functions of PACSINs, and then focus on the relevance of PACSINs in vivo. During development in various model organisms, PACSINs participate in diverse processes, such as neural crest cell development, gastrulation, laterality development and neuromuscular junction formation. In mouse, PACSIN2 regulates angiogenesis during retinal development and in human, PACSIN2 associates with monosomy and embryonic implantation. In adulthood, PACSIN1 has been extensively studied in the brain and shown to regulate neuromorphogenesis, receptor trafficking and synaptic plasticity. Several genetic studies suggest a role for PACSIN1 in the development of schizophrenia, which is also supported by the phenotype of mice depleted of PACSIN1. PACSIN2 plays an essential role in the maintenance of intestinal homeostasis and participates in kidney repair processes after injury. PACSIN3 is abundant in muscle tissue and necessary for caveolar biogenesis to create membrane reservoirs, thus controlling muscle function, and has been linked to certain genetic muscular disorders. The above examples illustrate the importance of PACSINs in diverse physiological or tissue repair processes in various organs, and associations to diseases when their functions are disturbed.

TMEM16F and dynamins control expansive plasma membrane reservoirs

Cells can expand their plasma membrane laterally by unfolding membrane undulations and by exocytosis. Here, we describe a third mechanism involving invaginations held shut by the membrane adapter, dynamin. Compartments open when Ca activates the lipid scramblase, TMEM16F, anionic phospholipids escape from the cytoplasmic monolayer in exchange for neutral lipids, and dynamins relax. Deletion of TMEM16F or dynamins blocks expansion, with loss of dynamin expression generating a maximally expanded basal plasma membrane state. Re-expression of dynamin2 or its GTPase-inactivated mutant, but not a lipid binding mutant, regenerates reserve compartments and rescues expansion. Dynamin2-GFP fusion proteins form punctae that rapidly dissipate from these compartments during TMEM16F activation. Newly exposed compartments extend deeply into the cytoplasm, lack numerous organellar markers, and remain closure-competent for many seconds. Without Ca, compartments open slowly when dynamins are sequestered by cytoplasmic dynamin antibodies or when scrambling is mimicked by neutralizing anionic phospholipids and supplementing neutral lipids. Activation of Ca-permeable mechanosensitive channels via cell swelling or channel agonists opens the compartments in parallel with phospholipid scrambling. Thus, dynamins and TMEM16F control large plasma membrane reserves that open in response to lateral membrane stress and Ca influx.

A \u2018dynamic adder model\u2019 for cell size homeostasis in Dictyostelium cells

After a cell divides into two daughter cells, the total cell surface area of the daughter cells should increase to the original size to maintain cell size homeostasis in a single cell cycle. Previously, three models have been proposed to explain the regulation of cell size homeostasis: sizer, timer, and adder models. Here, we precisely measured the total cell surface area of Dictyostelium cells in a whole cell cycle by using the agar-overlay method, which eliminated the influence of surface membrane reservoirs, such as microvilli and membrane wrinkles. The total cell surface area exponentially increased during interphase, slightly decreased at metaphase, and then increased by approximately 20% during cytokinesis. From the analysis of the added surface area, we concluded that the cell size was regulated by the adder or near-adder model in interphase. This adder model is not caused by a simple cell membrane addition, but is more dynamic due to the rapid cell membrane turnover. We propose a ‘dynamic adder model’ to explain cell size homeostasis in interphase.

Biomechanics of Neutrophil Tethers.

Leukocytes, including neutrophils, propelled by blood flow, can roll on inflamed endothelium using transient bonds between selectins and their ligands, and integrins and their ligands. When such receptor–ligand bonds last long enough, the leukocyte microvilli become extended and eventually form thin, 20 µm long tethers. Tether formation can be observed in blood vessels in vivo and in microfluidic flow chambers. Tethers can also be extracted using micropipette aspiration, biomembrane force probe, optical trap, or atomic force microscopy approaches. Here, we review the biomechanical properties of leukocyte tethers as gleaned from such measurements and discuss the advantages and disadvantages of each approach. We also review and discuss viscoelastic models that describe the dependence of tether formation on time, force, rate of loading, and cell activation. We close by emphasizing the need to combine experimental observations with quantitative models and computer simulations to understand how tether formation is affected by membrane tension, membrane reservoir, and interactions of the membrane with the cytoskeleton.

Cell Surface Area to Volume Relationship During Apoptosis and Apoptotic Body Formation.

Apoptosis is a programmed form of cell death culminating in packing cell content and corpse dismantling into membrane sealed vesicles called apoptotic bodies (ABs). Apoptotic bodies are engulfed and disposed by neighboring and immune system cells without eliciting a noxious inflammatory response, thus preventing sterile tissue damage. AB formation requires a total surface area larger than the apparent, initial cell's surface area. Apoptotic volume decrease (AVD) is a two-stage process leading to an isotonic, osmotic reduction in cell water content driven by net K+ and Cl- extrusion. In this article, the role of AVD is presented from a geometric point of view through the process of AB formation. AVD decisively contributes to (i) endowing the cell with the appropriate electrolytic environment for apoptotic execution; (ii) increasing the membrane surface area-to-volume ratio, along with the mobilization of membrane reservoirs (cell rounding, membrane folds and endosomal membranes), so that the cell corpse can be dismantled into ABs; and (iii) reducing plasmalemmal stretch, tension and stiffness, thus facilitating membrane bulging, blebbing and vesicle expansion ultimately leading to separation and release.

Functional implications of Pacsin2 localization in mast cells

Mast cell activation and degranulation involves rapid and dramatic reorganization of plasma membrane and cytoskeleton. We discovered that the protein Pacsin2 (syndapin II) localizes in distinct ring-like structures on resting mast cell plasma membranes. These rings surround areas of high F-actin and plasma membrane signals, resembling reservoirs. Activation through FCER1 or thapsigargin rapidly disassemble Pacsin2 rings and reservoirs, which reform when activation ceases. Localization of Pacsin2 to rings is dependent on the F-BAR domain region of the protein, which is responsible for plasma membrane bending and curvature sensing. Pacsin2 ring formation is anti-correlated with the activity of N-WASP, an Arp2/3-activating protein that promotes F-actin assembly and degranulation downstream of FCER1. Notably, N-WASP inhibition increases Pacsin2 localization at the plasma membrane. Due to its dynamic ring-like organization at membrane reservoirs and opposing relationship to N-WASP during cell activation, Pacsin2 may serve as a conduit between structural organization of the plasma membrane and downstream signaling to impact mast cell functions such as degranulation.

Cell surface topography controls phagocytosis and cell spreading: The membrane reservoir in neutrophils

Neutrophils exhibit rapid cell spreading and phagocytosis, both requiring a large apparent increase in the cell surface area. The wrinkled surface topography of these cells may provide the membrane reservoir for this. Here, the effects of manipulation of the neutrophil cell surface topography on phagocytosis and cell spreading were established. Chemical expansion of the plasma membrane or osmotic swelling had no effects. However, osmotic shrinking of neutrophils inhibited both cell spreading and phagocytosis. Triggering a Ca2+ signal in osmotically shrunk cells (by IP3 uncaging) evoked tubular blebs instead of full cell spreading. Phagocytosis was halted at the phagocytic cup stage by osmotic shrinking induced after the phagocytic Ca2+ signalling. Restoration of isotonicity was able to restore complete phagocytosis. These data thus provide evidence that the wrinkled neutrophil surface topography provides the membrane reservoir to increase the available cell surface area for phagocytosis and spreading by neutrophils.