A live cell imaging-based assay for tracking particle uptake by clathrin-mediated endocytosis.

A popular strategy for therapeutic delivery to cells and tissues is to encapsulate therapeutics inside particles that cells internalize via endocytosis. The efficacy of particle uptake by endocytosis is often studied in bulk using flow cytometry and Western blot analysis and confirmed using confocal microscopy. However, these techniques do not reveal the detailed dynamics of particle internalization and how the inherent heterogeneity of many types of particles may impact their endocytic uptake. Toward addressing these gaps, here we present a live-cell imaging-based method that utilizes total internal reflection fluorescence microscopy to track the uptake of a large ensemble of individual particles in parallel, as they interact with the cellular endocytic machinery. To analyze the resulting data, we employ an open-source tracking algorithm in combination with custom data filters. This analysis reveals the dynamic interactions between particles and endocytic structures, which determine the probability of particle uptake. In particular, our approach can be used to examine how variations in the physical properties of particles (size, targeting, rigidity), as well as heterogeneity within the particle population, impact endocytic uptake. These data impact the design of particles toward more selective and efficient delivery of therapeutics to cells.

Engineered mini-G proteins block the internalization of cognate GPCRs and disrupt downstream intracellular signaling.

Mini-G proteins are engineered, thermostable variants of Gα subunits designed to stabilize G protein-coupled receptors (GPCRs) in their active conformations. Because of their small size and ease of use, they are popular tools for assessing GPCR behaviors in cells, both as reporters of receptor coupling to Gα subtypes and for cellular assays to quantify compartmentalized signaling at various subcellular locations. Here, we report that overexpression of mini-G proteins with their cognate GPCRs disrupted GPCR endocytic trafficking and associated intracellular signaling. In cells expressing the Gαs-coupled GPCR glucagon-like peptide 1 receptor (GLP-1R), coexpression of mini-Gs, a mini-G protein derived from Gαs, blocked ß-arrestin 2 recruitment and receptor internalization and disrupted endosomal GLP-1R signaling. These effects did not involve changes in receptor phosphorylation or lipid nanodomain segregation. Moreover, we found that mini-G proteins derived from Gαi and Gαq also inhibited the internalization of GPCRs that couple to them. Finally, we developed an alternative intracellular signaling assay for GLP-1R using a nanobody specific for active Gαs:GPCR complexes (Nb37) that did not affect GLP-1R internalization. Our results have important implications for designing methods to assess intracellular GPCR signaling.

Endocytosis in anaerobic parasitic protists.

The incorporation of different molecules by eukaryotic cells occurs through endocytosis, which is critical to the cell's survival and ability to reproduce. Although this process has been studied in greater detail in mammalian and yeast cells, several groups working with pathogenic protists have made relevant contributions. This review analysed the most relevant data on the endocytic process in anaerobic protists (Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis, and Tritrichomonas foetus). Many protozoa can exert endocytic activity across their entire surface and do so with great intensity, as with E. histolytica. The available data on the endocytic pathway and the participation of PI-3 kinase, Rab, and Rho molecular complexes is reviewed from a historical perspective.

Roles of membrane mechanics-mediated feedback in membrane traffic.

Synthesizing the recent progresses, we present our perspectives on how local modulations of membrane curvature, tension, and bending energy define the feedback controls over membrane traffic processes. We speculate the potential mechanisms of, and the control logic behind, the different membrane mechanics-mediated feedback in endocytosis and exo-endocytosis coupling. We elaborate the path forward with the open questions for theoretical considerations and the grand challenges for experimental validations.

Physiological roles of endocytosis and presynaptic scaffold in vesicle replenishment at fast and slow central synapses.

After exocytosis, release sites are cleared of vesicular residues to replenish with transmitter-filled vesicles. Endocytic and scaffold proteins are thought to underlie this site-clearance mechanism. However, the physiological significance of this mechanism at diverse mammalian central synapses remains unknown. Here, we tested this in a physiologically optimized condition using action potential evoked EPSCs at fast calyx synapse and relatively slow hippocampal CA1 synapse, in post-hearing mice brain slices at 37°C and in 1.3 mM [Ca2+]. Pharmacological block of endocytosis enhanced synaptic depression at the calyx synapse, whereas it attenuated synaptic facilitation at the hippocampal synapse. Block of scaffold protein activity likewise enhanced synaptic depression at the calyx but had no effect at the hippocampal synapse. At the fast calyx synapse, block of endocytosis or scaffold protein activity significantly enhanced synaptic depression as early as 10 ms after the stimulation onset. Unlike previous reports, neither endocytic blockers nor scaffold protein inhibitors prolonged the recovery from short-term depression. We conclude that the release-site clearance by endocytosis can be a universal phenomenon supporting vesicle replenishment at both fast and slow synapses, whereas the presynaptic scaffold mechanism likely plays a specialized role in vesicle replenishment predominantly at fast synapses.

G protein-coupled receptor endocytosis generates spatiotemporal bias in ß-arrestin signaling.

The stabilization of different active conformations of G protein-coupled receptors is thought to underlie the varying efficacies of biased and balanced agonists. Here, profiling the activation of signal transducers by angiotensin II type 1 receptor (AT1R) agonists revealed that the extent and kinetics of ß-arrestin binding exhibited substantial ligand-dependent differences, which were lost when receptor internalization was inhibited. When AT1R endocytosis was prevented, even weak partial agonists of the ß-arrestin pathway acted as full or near-full agonists, suggesting that receptor conformation did not exclusively determine ß-arrestin recruitment. The ligand-dependent variance in ß-arrestin translocation was much larger at endosomes than at the plasma membrane, showing that ligand efficacy in the ß-arrestin pathway was spatiotemporally determined. Experimental investigations and mathematical modeling demonstrated how multiple factors concurrently shaped the effects of agonists on endosomal receptor-ß-arrestin binding and thus determined the extent of functional selectivity. Ligand dissociation rate and G protein activity had particularly strong, internalization-dependent effects on the receptor-ß-arrestin interaction. We also showed that endocytosis regulated the agonist efficacies of two other receptors with sustained ß-arrestin binding: the V2 vasopressin receptor and a mutant ß2-adrenergic receptor. In the absence of endocytosis, the agonist-dependent variance in ß-arrestin2 binding was markedly diminished. Our results suggest that endocytosis determines the spatiotemporal bias in GPCR signaling and can aid in the development of more efficacious, functionally selective compounds.

Generation of a bloodstream form Trypanosoma brucei double glycosyltransferase null mutant competent in receptor-mediated endocytosis of transferrin.

The bloodstream form of Trypanosoma brucei expresses large poly-N-acetyllactosamine (pNAL) chains on complex N-glycans of a subset of glycoproteins. It has been hypothesised that pNAL may be required for receptor-mediated endocytosis. African trypanosomes contain a unique family of glycosyltransferases, the GT67 family. Two of these, TbGT10 and TbGT8, have been shown to be involved in pNAL biosynthesis in bloodstream form Trypanosoma brucei, raising the possibility that deleting both enzymes simultaneously might abolish pNAL biosynthesis and provide clues to pNAL function and/or essentiality. In this paper, we describe the creation of a TbGT10 null mutant containing a single TbGT8 allele that can be excised upon the addition of rapamycin and, from that, a TbGT10 and TbGT8 double null mutant. These mutants were analysed by lectin blotting, glycopeptide methylation linkage analysis and flow cytometry. The data show that the mutants are defective, but not abrogated, in pNAL synthesis, suggesting that other GT67 family members can compensate to some degree for loss of TbGT10 and TbGT8. Despite there being residual pNAL synthesis in these mutants, certain glycoproteins appear to be particularly affected. These include the lysosomal CBP1B serine carboxypeptidase, cell surface ESAG2 and the ESAG6 subunit of the essential parasite transferrin receptor (TfR). The pNAL deficient TfR in the mutants continued to function normally with respect to protein stability, transferrin binding, receptor mediated endocytosis of transferrin and subcellular localisation. Further the pNAL deficient mutants were as viable as wild type parasites in vitro and in in vivo mouse infection experiments. Although we were able to reproduce the inhibition of transferrin uptake with high concentrations of pNAL structural analogues (N-acetylchito-oligosaccharides), this effect disappeared at lower concentrations that still inhibited tomato lectin uptake, i.e., at concentrations able to outcompete lectin-pNAL binding. Based on these findings, we recommend revision of the pNAL-dependent receptor mediated endocytosis hypothesis.

Renal handling of albumin in rats with early stage diabetes: A theoretical analysis.

In early diabetic nephropathy (DN), recent studies have shown that albuminuria stems mostly from alterations in tubular function rather than from glomerular damage. Several factors in DN, including hyperfiltration, hypertrophy and reduced abundance of the albumin receptors megalin and cubilin, affect albumin endocytosis in the proximal tubule (PT). To assess their respective contribution, we developed a model of albumin handling in the rat PT that couples the transport of albumin to that of water and solutes. Our simulations suggest that, under basal conditions, ∼75% of albumin is retrieved in the S1 segment. The model predicts negligible uptake in S3, as observed experimentally. It also accurately predicts the impact of acute hyperglycaemia on urinary albumin excretion. Simulations reproduce observed increases in albumin excretion in early DN by considering the combined effects of increased glomerular filtration rate (GFR), osmotic diuresis, hypertrophy, and megalin and cubilin downregulation, without stipulating changes in glomerular permselectivity. The results indicate that in isolation, glucose-elicited osmotic diuresis and glucose transporter upregulation raise albumin excretion only slightly. Enlargement of PT diameter not only augments uptake via surface area expansion, but also reduces fluid velocity and thus shear stress-induced stimulation of endocytosis. Overall, our model predicts that downregulation of megalin and cubilin and hyperfiltration both contribute significantly to increasing albumin excretion in rats with early-stage diabetes. The results also suggest that acute sodium-glucose cotransporter 2 inhibition lowers albumin excretion only if GFR decreases sufficiently, and that angiotensin II receptor blockers mitigate urinary albumin loss in early DN in large part by upregulating albumin receptor abundance. KEY POINTS: The urinary excretion of albumin is increased in early diabetic nephropathy (DN). It is difficult to experimentally disentangle the multiple factors that affect the renal handling of albumin in DN. We developed a mathematical model of albumin transport in the rat proximal tubule (PT) to examine the impact of elevated plasma glucose, hyperfiltration, PT hypertrophy and reduced abundance of albumin receptors on albumin uptake and excretion in DN. Our model predicts that glucose-elicited osmotic diuresis per se raises albumin excretion only slightly. Conversely, increases in PT diameter and length favour reduced albumin excretion. Our results suggest that downregulation of the receptors megalin and cubilin in PT cells and hyperfiltration both contribute significantly to increasing albumin excretion in DN. The model helps to better understand the mechanisms underlying urinary loss of albumin in early-stage diabetes, and the impact of specific treatments thereupon.

Intermittent hypoxia training enhances Aß endocytosis by plaque associated microglia via VPS35-dependent TREM2 recycling in murine Alzheimer's disease.

BACKGROUND: Beta-amyloid (Aß) deposition in the brain parenchyma is a crucial initiating step in the amyloid cascade hypothesis of Alzheimer's disease (AD) pathology. Furthermore, dysfunction of plaque-associated microglia, also known as disease-associated microglia (DAM) has been reported to accelerate Aß deposition and cognitive impairment. Our previous research demonstrated that intermittent hypoxia training (IHT) improved AD pathology by upregulating autophagy in DAM, thereby enhancing oligomeric Aß (oAß) clearance. Considering that oAß internalization is the initial stage of oAß clearance, this study focused on the IHT mechanism involved in upregulating Aß uptake by DAM. METHODS: IHT was administered to 8-month-old APP/PS1 mice or 6-month-old microglial vacuolar protein sorting 35 (VPS35) knockout mice in APP/PS1 background (MG VPS35 KO: APP/PS1) for 28 days. After the IHT, the spatial learning-memory capacity of the mice was assessed. Additionally, AD pathology was determined by estimating the nerve fiber and synapse density, Aß plaque deposition, and Aß load in the brain. A model of Aß-exposed microglia was constructed and treated with IHT to explore the related mechanism. Finally, triggering receptor expressed on myeloid cells 2 (TREM2) intracellular recycling and Aß internalization were measured using a fluorescence tracing technique. RESULTS: Our results showed that IHT ameliorated cognitive function and Aß pathology. In particular, IHT enhanced Aß endocytosis by augmenting the intracellular transport function of microglial TREM2, thereby contributing to Aß clearance. Furthermore, IHT specifically upregulated VPS35 in DAM, the primary cause for the enhanced intracellular recycling of TREM2. IHT lost ameliorative effect on Aß pathology in MG VPS35 KO: APP/PS1 mice brain. Lastly, the IHT mechanism of VPS35 upregulation in DAM was mediated by the transcriptional regulation of VPS35 by transcription factor EB (TFEB). CONCLUSION: IHT enhances Aß endocytosis in DAM by upregulating VPS35-dependent TREM2 recycling, thereby facilitating oAß clearance and mitigation of Aß pathology. Moreover, the transcriptional regulation of VPS35 by TFEB demonstrates a close link between endocytosis and autophagy in microglia. Our study further elucidates the IHT mechanism in improving AD pathology and provides evidence supporting the potential application of IHT as a complementary therapy for AD.

Phospholipid Scramblase 1 Controls Efficient Neurotransmission and Synaptic Vesicle Retrieval at Cerebellar Synapses.

Phospholipids (PLs) are asymmetrically distributed at the plasma membrane. This asymmetric lipid distribution is transiently altered during calcium-regulated exocytosis, but the impact of this transient remodeling on presynaptic function is currently unknown. As phospholipid scramblase 1 (PLSCR1) randomizes PL distribution between the two leaflets of the plasma membrane in response to calcium activation, we set out to determine its role in neurotransmission. We report here that PLSCR1 is expressed in cerebellar granule cells (GrCs) and that PLSCR1-dependent phosphatidylserine egress occurred at synapses in response to neuron stimulation. Synaptic transmission is impaired at GrC Plscr1 -/- synapses, and both PS egress and synaptic vesicle (SV) endocytosis are inhibited in Plscr1 -/- cultured neurons from male and female mice, demonstrating that PLSCR1 controls PL asymmetry remodeling and SV retrieval following neurotransmitter release. Altogether, our data reveal a novel key role for PLSCR1 in SV recycling and provide the first evidence that PL scrambling at the plasma membrane is a prerequisite for optimal presynaptic performance.

Difference in Endocytosis Pathways Used by Differentiated Versus Nondifferentiated Epithelial Caco-2 Cells to Internalize Nanosized Particles.

Understanding the internalization of nanosized particles by mucosal epithelial cells is essential in a number of areas including viral entry at mucosal surfaces, nanoplastic pollution, as well as design and development of nanotechnology-type medicines. Here, we report our comparative study on pathways of cellular internalization in epithelial Caco-2 cells cultured in vitro as either a polarized, differentiated cell layer or as nonpolarized, nondifferentiated cells. The study reveals a number of differences in the extent that endocytic processes are used by cells, depending on their differentiation status and the nature of applied nanoparticles. In polarized cells, actin-driven and dynamin-independent macropinocytosis plays a prominent role in the internalization of both positively and negatively charged nanoparticles, contrary to its modest contribution in nonpolarized cells. Clathrin-mediated cellular entry plays a prominent role in the endocytosis of positive nanoparticles and cholesterol inhibition in negative nanoparticles. However, in nonpolarized cells, dynamin-dependent endocytosis is a major pathway in the internalization of both positive and negative nanoparticles. Cholesterol depletion affects both nonpolarized and polarized cells' internalization of positive and negative nanoparticles, which, in addition to the effect of cholesterol-binding inhibitors on the internalization of negative nanoparticles, indicates the importance of membrane cholesterol in endocytosis. The data collectively provide a new contribution to understanding endocytic pathways in epithelial cells, particularly pointing to the importance of the cell differentiation stage and the nature of the cargo.

Endolysosomal trapping of therapeutics and endosomal escape strategies.

Internalizing therapeutic molecules or genes into cells and safely delivering them to the target tissue where they can perform the intended tasks is one of the key characteristics of the smart gene/drug delivery vector. Despite much research in this field, endosomal escape continues to be a significant obstacle to the development of effective gene/drug delivery systems. In this review, we discuss in depth the several types of endocytic pathways involved in the endolysosomal trapping of therapeutic agents. In addition, we describe numerous mechanisms involved in nanoparticle endosomal escape. Furthermore, many other techniques are employed to increase endosomal escape to minimize entrapment of therapeutic compounds within endolysosomes, which have been reviewed at length in this study.

Optimization of the fluorogen-activating protein tag for quantitative protein trafficking and colocalization studies in S. cerevisiae.

Spatial and temporal tracking of fluorescent proteins (FPs) in live cells permits visualization of proteome remodeling in response to extracellular cues. Historically, protein dynamics during trafficking have been visualized using constitutively active FPs fused to proteins of interest. While powerful, such FPs label all cellular pools of a protein, potentially masking the dynamics of select subpopulations. To help study protein subpopulations, bioconjugate tags, including the fluorogen activation proteins (FAPs), were developed. FAPs are comprised of two components: a single-chain antibody (SCA) fused to the protein of interest and a malachite-green (MG) derivative, which fluoresces only when bound to the SCA. Importantly, the MG derivatives can be either cell-permeant or -impermeant, thus permitting isolated detection of SCA-tagged proteins at the cell surface and facilitating quantitative endocytic measures. To expand FAP use in yeast, we optimized the SCA for yeast expression, created FAP-tagging plasmids, and generated FAP-tagged organelle markers. To demonstrate FAP efficacy, we coupled the SCA to the yeast G-protein coupled receptor Ste3. We measured Ste3 endocytic dynamics in response to pheromone and characterized cis- and trans-acting regulators of Ste3. Our work significantly expands FAP technology for varied applications in S. cerevisiae.

AP2A2 mutation and defective endocytosis in a Malian family with hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) comprises a large group of neurogenetic disorders characterized by progressive lower extremity spasticity. Neurological evaluation and genetic testing were completed in a Malian family with early-onset HSP. Three children with unaffected consanguineous parents presented with symptoms consistent with childhood-onset complicated HSP. Neurological evaluation found lower limb weakness, spasticity, dysarthria, seizures, and intellectual disability. Brain MRI showed corpus callosum thinning with cortical and spinal cord atrophy, and an EEG detected slow background in the index patient. Whole exome sequencing identified a homozygous missense variant in the adaptor protein (AP) complex 2 alpha-2 subunit (AP2A2) gene. Western blot analysis showed reduced levels of AP2A2 in patient-iPSC derived neuronal cells. Endocytosis of transferrin receptor (TfR) was decreased in patient-derived neurons. In addition, we observed increased axon initial segment length in patient-derived neurons. Xenopus tropicalis tadpoles with ap2a2 knockout showed cerebral edema and progressive seizures. Immunoprecipitation of the mutant human AP-2-appendage alpha-C construct showed defective binding to accessory proteins. We report AP2A2 as a novel genetic entity associated with HSP and provide functional data in patient-derived neuron cells and a frog model. These findings expand our understanding of the mechanism of HSP and improve the genetic diagnosis of this condition.

Metabolic regulation of single synaptic vesicle exo- and endocytosis in hippocampal synapses.

Glucose has long been considered a primary energy source for synaptic function. However, it remains unclear to what extent alternative fuels, such as lactate/pyruvate, contribute to powering synaptic transmission. By detecting individual release events in hippocampal synapses, we find that mitochondrial ATP production regulates basal vesicle release probability and release location within the active zone (AZ), evoked by single action potentials. Mitochondrial inhibition shifts vesicle release closer to the AZ center and alters the efficiency of vesicle retrieval by increasing the occurrence of ultrafast endocytosis. Furthermore, we uncover that terminals can use oxidative fuels to maintain the vesicle cycle during trains of activity. Mitochondria are sparsely distributed along hippocampal axons, and we find that terminals containing mitochondria display enhanced vesicle release and reuptake during high-frequency trains. Our findings suggest that mitochondria not only regulate several fundamental features of synaptic transmission but may also contribute to modulation of short-term synaptic plasticity.

Vesicle and reaction-diffusion hybrid modeling with STEPS.

Vesicles carry out many essential functions within cells through the processes of endocytosis, exocytosis, and passive and active transport. This includes transporting and delivering molecules between different parts of the cell, and storing and releasing neurotransmitters in neurons. To date, computational simulation of these key biological players has been rather limited and has not advanced at the same pace as other aspects of cell modeling, restricting the realism of computational models. We describe a general vesicle modeling tool that has been designed for wide application to a variety of cell models, implemented within our software STochastic Engine for Pathway Simulation (STEPS), a stochastic reaction-diffusion simulator that supports realistic reconstructions of cell tissue in tetrahedral meshes. The implementation is validated in an extensive test suite, parallel performance is demonstrated in a realistic synaptic bouton model, and example models are visualized in a Blender extension module.

The mechanism of mitochondrial autophagy regulating Clathrin-mediated endocytosis in epilepsy.

OBJECTIVE: The objective of this study is to determine whether inhibition of mitophagy affects seizures through Clathrin-mediated endocytosis (CME). METHODS: Pentylenetetrazol (PTZ) was intraperitoneally injected daily to establish a chronic PTZ-kindled seizure. The Western blot (WB) was used to compare the differences in Parkin protein expression between the epilepsy group and the control group. Immunofluorescence was used to detect the expression of MitoTracker and LysoTracker. Transferrin-Alexa488 (Tf-A488) was injected into the hippocampus of mice. We evaluated the effect of 3-methyladenine (3-MA) on epilepsy behavior through observation in PTZ-kindled models. RESULTS: The methylated derivative of adenine, known as 3-MA, has been extensively utilized in the field of autophagy research. The transferrin protein is internalized from the extracellular environment into the intracellular space via the CME pathway. Tf-A488 uses a fluorescent marker to track CME. Western blot showed that the expression of Parkin was significantly increased in the PTZ-kindled model (p < 0.05), while 3-MA could reduce the expression (p < 0.05). The fluorescence uptake of MitoTracker and LysoTracker was increased in the primary cultured neurons induced by magnesium-free extracellular fluid (p < 0.05); the fluorescence uptake of Tf-A488 was significantly decreased in the 3-MA group compared with the control group (p < 0.05). Following hippocampal injection of Tf-A488, both the epilepsy group and the 3-MA group exhibited decreased fluorescence uptake, with a more pronounced effect observed in the 3-MA group. Inhibition of mitophagy by 3-MA from day 3 to day 9 progressively exacerbated seizure severity and shortened latency. SIGNIFICANCE: It is speculated that the aggravation of seizures by 3-MA may be related to the failure to remove damaged mitochondria in time and effectively after inhibiting mitochondrial autophagy, affecting the vesicle endocytosis function of CME and increasing the susceptibility to epilepsy. SUMMARY: Abnormal mitophagy was observed in a chronic pentylenetetrazol-induced seizure model and a Mg2+-free-induced spontaneous recurrent epileptiform discharge model. A fluorescent transferrin marker was utilized to track clathrin-mediated endocytosis. Using an autophagy inhibitor (3-methyladenine) on primary cultured neurons, we discovered that inhibition of autophagy led to a reduction in fluorescent transferrin uptake, while impairing clathrin-mediated endocytosis function mediated by mitophagy. Finally, we examined the effects of 3-methyladenine in an animal model of seizures showing that it exacerbated seizure severity. Ultimately, this study provides insights into potential mechanisms through which mitophagy regulates clathrin-mediated endocytosis in epilepsy.

Photoswitchable Endocytosis of Biomolecular Condensates in Giant Vesicles.

Interactions between membranes and biomolecular condensates can give rise to complex phenomena such as wetting transitions, mutual remodeling, and endocytosis. In this study, light-triggered manipulation of condensate engulfment is demonstrated using giant vesicles containing photoswitchable lipids. UV irradiation increases the membrane area, which can be stored in nanotubes. When in contact with a condensate droplet, the UV light triggers rapid condensate endocytosis, which can be reverted by blue light. The affinity of the protein-rich condensates to the membrane and the reversibility of the engulfment processes is quantified from confocal microscopy images. The degree of photo-induced engulfment, whether partial or complete, depends on the vesicle excess area and the relative sizes of vesicles and condensates. Theoretical estimates suggest that utilizing the light-induced excess area to increase the vesicle-condensate adhesion interface is energetically more favorable than the energy gain from folding the membrane into invaginations and tubes. The overall findings demonstrate that membrane-condensate interactions can be easily and quickly modulated via light, providing a versatile system for building platforms to control cellular events and design intelligent drug delivery systems for cell repair.

Disrupting cell wall integrity impacts endomembrane trafficking to promote secretion over endocytic trafficking.

The plant cell wall provides a strong yet flexible barrier to protect cells from the external environment. Modifications of the cell wall, either during development or under stress conditions, can induce cell wall integrity responses and ultimately lead to alterations in gene expression, hormone production, and cell wall composition. These changes in cell wall composition presumably require remodelling of the secretory pathway to facilitate synthesis and secretion of cell wall components and cell wall synthesis/remodelling enzymes from the Golgi apparatus. Here, we used a combination of live-cell confocal imaging and transmission electron microscopy to examine the short-term and constitutive impact of isoxaben, which reduces cellulose biosynthesis, and Driselase, a cocktail of cell-wall-degrading fungal enzymes, on cellular processes during cell wall integrity responses in Arabidopsis. We show that both treatments altered organelle morphology and triggered rebalancing of the secretory pathway to promote secretion while reducing endocytic trafficking. The actin cytoskeleton was less dynamic following cell wall modification, and organelle movement was reduced. These results demonstrate active remodelling of the endomembrane system and actin cytoskeleton following changes to the cell wall.

The types and numbers of kinesins and dyneins transporting endocytic cargoes modulate their motility and response to tau.

Organelles and vesicular cargoes are transported by teams of kinesin and dynein motors along microtubules. We isolated endocytic organelles from cells at different stages of maturation and reconstituted their motility along microtubules in vitro. We asked how the sets of motors transporting a cargo determine its motility and response to the microtubule-associated protein tau. Here, we find that phagosomes move in both directions along microtubules, but the directional bias changes during maturation. Early phagosomes exhibit retrograde-biased transport while late phagosomes are directionally unbiased. Correspondingly, early and late phagosomes are bound by different numbers and combinations of kinesins-1, -2, -3, and dynein. Tau stabilizes microtubules and directs transport within neurons. While single-molecule studies show that tau differentially regulates the motility of kinesins and dynein in vitro, less is known about its role in modulating the trafficking of endogenous cargoes transported by their native teams of motors. Previous studies showed that tau preferentially inhibits kinesin motors, which biases late phagosome transport towards the microtubule minus-end. Here, we show that tau strongly inhibits long-range, dynein-mediated motility of early phagosomes. Tau reduces forces generated by teams of dynein motors on early phagosomes and accelerates dynein unbinding under load. Thus, cargoes differentially respond to tau, where dynein complexes on early phagosomes are more sensitive to tau inhibition than those on late phagosomes. Mathematical modeling further explains how small changes in the number of kinesins and dynein on cargoes impact the net directionality but also that cargoes with different sets of motors respond differently to tau.