Matthias Garten

All Publications

• Structured to conquer: transport across the Plasmodium parasitophorous vacuole CURRENT OPINION IN MICROBIOLOGY Garten, M., Beck, J. R. 2021; 63: 181-188

  Abstract

  During the vertebrate stage of the Plasmodium life cycle, obligate intracellular malaria parasites establish a vacuolar niche for replication, first within host hepatocytes at the pre-patent liver-stage and subsequently in erythrocytes during the pathogenic blood-stage. Survival in this protective microenvironment requires diverse transport mechanisms that enable the parasite to transcend the vacuolar barrier. Effector proteins exported out of the vacuole modify the erythrocyte membrane, increasing access to serum nutrients which then cross the vacuole membrane through a nutrient-permeable channel, supporting rapid parasite growth. This review highlights the most recent insights into the organization of the parasite vacuole to facilitate the solute, lipid and effector protein trafficking that establishes a nutrition pipeline in the terminally differentiated, organelle-free red blood cell.

  View details for DOI 10.1016/j.mib.2021.07.010

  View details for Web of Science ID 000701810100025

  View details for PubMedID 34375857

  View details for PubMedCentralID PMC8463430

• Contacting domains segregate a lipid transporter from a solute transporter in the malarial host-parasite interface NATURE COMMUNICATIONS Garten, M., Beck, J. R., Roth, R., Tenkova-Heuser, T., Heuser, J., Istvan, E. S., Bleck, C. E., Goldberg, D. E., Zimmerberg, J. 2020; 11 (1): 3825

  Abstract

  The malaria parasite interfaces with its host erythrocyte (RBC) using a unique organelle, the parasitophorous vacuole (PV). The mechanism(s) are obscure by which its limiting membrane, the parasitophorous vacuolar membrane (PVM), collaborates with the parasite plasma membrane (PPM) to support the transport of proteins, lipids, nutrients, and metabolites between the cytoplasm of the parasite and the cytoplasm of the RBC. Here, we demonstrate that the PV has structure characterized by micrometer-sized regions of especially close apposition between the PVM and the PPM. To determine if these contact sites are involved in any sort of transport, we localize the PVM nutrient-permeable and protein export channel EXP2, as well as the PPM lipid transporter PfNCR1. We find that EXP2 is excluded from, but PfNCR1 is included within these regions of close apposition. We conclude that the host-parasite interface is structured to segregate those transporters of hydrophilic and hydrophobic substrates.

  View details for DOI 10.1038/s41467-020-17506-9

  View details for Web of Science ID 000560076500010

  View details for PubMedID 32732874

  View details for PubMedCentralID PMC7393353

• EXP1 is critical for nutrient uptake across the parasitophorous vacuole membrane of malaria parasites PLOS BIOLOGY Mesen-Ramirez, P., Bergmann, B., Thuy Tuyen Tran, Garten, M., Staecker, J., Naranjo-Prado, I., Hoehn, K., Zimmerberg, J., Spielmann, T. 2019; 17 (9): e3000473

  Abstract

  Intracellular malaria parasites grow in a vacuole delimited by the parasitophorous vacuolar membrane (PVM). This membrane fulfils critical roles for survival of the parasite in its intracellular niche such as in protein export and nutrient acquisition. Using a conditional knockout (KO), we here demonstrate that the abundant integral PVM protein exported protein 1 (EXP1) is essential for parasite survival but that this is independent of its previously postulated function as a glutathione S-transferase (GST). Patch-clamp experiments indicated that EXP1 is critical for the nutrient-permeable channel activity at the PVM. Loss of EXP1 abolished the correct localisation of EXP2, a pore-forming protein required for the nutrient-permeable channel activity and protein export at the PVM. Unexpectedly, loss of EXP1 affected only the nutrient-permeable channel activity of the PVM but not protein export. Parasites with low levels of EXP1 became hypersensitive to low nutrient conditions, indicating that EXP1 indeed is needed for nutrient uptake and experimentally confirming the long-standing hypothesis that the channel activity measured at the PVM is required for parasite nutrient acquisition. Hence, EXP1 is specifically required for the functional expression of EXP2 as the nutrient-permeable channel and is critical for the metabolite supply of malaria parasites.

  View details for DOI 10.1371/journal.pbio.3000473

  View details for Web of Science ID 000496470000028

  View details for PubMedID 31568532

  View details for PubMedCentralID PMC6786648

• Rounding precedes rupture and breakdown of vacuolar membranes minutes before malaria parasite egress from erythrocytes. Cellular microbiology Glushakova, S., Beck, J. R., Garten, M., Busse, B. L., Nasamu, A. S., Tenkova-Heuser, T., Heuser, J., Goldberg, D. E., Zimmerberg, J. 2018; 20 (10): e12868

  Abstract

  Because Plasmodium falciparum replicates inside of a parasitophorous vacuole (PV) within a human erythrocyte, parasite egress requires the rupture of two limiting membranes. Parasite Ca2+ , kinases, and proteases contribute to efficient egress; their coordination in space and time is not known. Here, the kinetics of parasite egress were linked to specific steps with specific compartment markers, using live-cell microscopy of parasites expressing PV-targeted fluorescent proteins, and specific egress inhibitors. Several minutes before egress, under control of parasite [Ca2+ ]i , the PV began rounding. Then after ~1.5 min, under control of PfPKG and SUB1, there was abrupt rupture of the PV membrane and release of vacuolar contents. Over the next ~6 min, there was progressive vacuolar membrane deterioration simultaneous with erythrocyte membrane distortion, lasting until the final minute of the egress programme when newly formed parasites mobilised and erythrocyte membranes permeabilised and then ruptured-a dramatic finale to the parasite cycle of replication.

  View details for DOI 10.1111/cmi.12868

  View details for PubMedID 29900649

  View details for PubMedCentralID PMC6368063

• EXP2 is a nutrient-permeable channel in the vacuolar membrane of Plasmodium and is essential for protein export via PTEX NATURE MICROBIOLOGY Garten, M., Nasamu, A. S., Niles, J. C., Zimmerberg, J., Goldberg, D. E., Beck, J. R. 2018; 3 (10): 1090-+

  Abstract

  Intraerythrocytic malaria parasites reside within a parasitophorous vacuolar membrane (PVM) generated during host cell invasion1. Erythrocyte remodelling and parasite metabolism require the export of effector proteins and transport of small molecules across this barrier between the parasite surface and host cell cytosol2,3. Protein export across the PVM is accomplished by the Plasmodium translocon of exported proteins (PTEX) consisting of three core proteins, the AAA+ ATPase HSP101 and two additional proteins known as PTEX150 and EXP24. Inactivation of HSP101 and PTEX150 arrests protein export across the PVM5,6, but the contribution of EXP2 to parasite biology is not well understood7. A nutrient permeable channel in the PVM has also been characterized electrophysiologically, but its molecular identity is unknown8,9. Here, using regulated gene expression, mutagenesis and cell-attached patch-clamp measurements, we show that EXP2, the putative membrane-spanning channel of PTEX4,10-14, serves dual roles as a protein-conducting channel in the context of PTEX and as a channel able to facilitate nutrient passage across the PVM independent of HSP101. Our data suggest a dual functionality for a channel operating in its endogenous context.

  View details for DOI 10.1038/s41564-018-0222-7

  View details for Web of Science ID 000448228400006

  View details for PubMedID 30150733

  View details for PubMedCentralID PMC6158082

• Exploitation of a newly-identified entry pathway into the malaria parasite-infected erythrocyte to inhibit parasite egress SCIENTIFIC REPORTS Glushakova, S., Busse, B. L., Garten, M., Beck, J. R., Fairhurst, R. M., Goldberg, D. E., Zimmerberg, J. 2017; 7: 12250

  Abstract

  While many parasites develop within host cells to avoid antibody responses and to utilize host cytoplasmic resources, elaborate egress processes have evolved to minimize the time between escaping and invading the next cell. In human erythrocytes, malaria parasites perforate their enclosing erythrocyte membrane shortly before egress. Here, we show that these pores clearly function as an entry pathway into infected erythrocytes for compounds that inhibit parasite egress. The natural glycosaminoglycan heparin surprisingly inhibited malaria parasite egress, trapping merozoites within infected erythrocytes. Labeled heparin neither bound to nor translocated through the intact erythrocyte membrane during parasite development, but fluxed into erythrocytes at the last minute of the parasite lifecycle. This short encounter was sufficient to significantly inhibit parasite egress and dispersion. Heparin blocks egress by interacting with both the surface of intra-erythrocytic merozoites and the inner aspect of erythrocyte membranes, preventing the rupture of infected erythrocytes but not parasitophorous vacuoles, and independently interfering with merozoite disaggregation. Since this action of heparin recapitulates that of neutralizing antibodies, membrane perforation presents a brief opportunity for a new strategy to inhibit parasite egress and replication.

  View details for DOI 10.1038/s41598-017-12258-x

  View details for Web of Science ID 000411648500024

  View details for PubMedID 28947749

  View details for PubMedCentralID PMC5612957

• Whole-GUV patch-clamping PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Garten, M., Mosgaard, L. D., Bornschloegl, T., Dieudonne, S., Bassereau, P., Toombes, G. S. 2017; 114 (2): 328-333

  Abstract

  Studying how the membrane modulates ion channel and transporter activity is challenging because cells actively regulate membrane properties, whereas existing in vitro systems have limitations, such as residual solvent and unphysiologically high membrane tension. Cell-sized giant unilamellar vesicles (GUVs) would be ideal for in vitro electrophysiology, but efforts to measure the membrane current of intact GUVs have been unsuccessful. In this work, two challenges for obtaining the "whole-GUV" patch-clamp configuration were identified and resolved. First, unless the patch pipette and GUV pressures are precisely matched in the GUV-attached configuration, breaking the patch membrane also ruptures the GUV. Second, GUVs shrink irreversibly because the membrane/glass adhesion creating the high-resistance seal (>1 GΩ) continuously pulls membrane into the pipette. In contrast, for cell-derived giant plasma membrane vesicles (GPMVs), breaking the patch membrane allows the GPMV contents to passivate the pipette surface, thereby dynamically blocking membrane spreading in the whole-GMPV mode. To mimic this dynamic passivation mechanism, beta-casein was encapsulated into GUVs, yielding a stable, high-resistance, whole-GUV configuration for a range of membrane compositions. Specific membrane capacitance measurements confirmed that the membranes were truly solvent-free and that membrane tension could be controlled over a physiological range. Finally, the potential for ion transport studies was tested using the model ion channel, gramicidin, and voltage-clamp fluorometry measurements were performed with a voltage-dependent fluorophore/quencher pair. Whole-GUV patch-clamping allows ion transport and other voltage-dependent processes to be studied while controlling membrane composition, tension, and shape.

  View details for DOI 10.1073/pnas.1609142114

  View details for Web of Science ID 000391439300047

  View details for PubMedID 28003462

  View details for PubMedCentralID PMC5240670

• Absence of the ER Cation Channel TMEM38B/TRIC-B Disrupts Intracellular Calcium Homeostasis and Dysregulates Collagen Synthesis in Recessive Osteogenesis Imperfecta PLOS GENETICS Cabral, W. A., Ishikawa, M., Garten, M., Makareeva, E. N., Sargent, B. M., Weis, M., Barnes, A. M., Webb, E. A., Shaw, N. J., Ala-Kokko, L., Lacbawan, F. L., Hogler, W., Leikin, S., Blank, P. S., Zimmerberg, J., Eyre, D. R., Yamada, Y., Marini, J. C. 2016; 12 (7): e1006156

  Abstract

  Recessive osteogenesis imperfecta (OI) is caused by defects in proteins involved in post-translational interactions with type I collagen. Recently, a novel form of moderately severe OI caused by null mutations in TMEM38B was identified. TMEM38B encodes the ER membrane monovalent cation channel, TRIC-B, proposed to counterbalance IP3R-mediated Ca2+ release from intracellular stores. The molecular mechanisms by which TMEM38B mutations cause OI are unknown. We identified 3 probands with recessive defects in TMEM38B. TRIC-B protein is undetectable in proband fibroblasts and osteoblasts, although reduced TMEM38B transcripts are present. TRIC-B deficiency causes impaired release of ER luminal Ca2+, associated with deficient store-operated calcium entry, although SERCA and IP3R have normal stability. Notably, steady state ER Ca2+ is unchanged in TRIC-B deficiency, supporting a role for TRIC-B in the kinetics of ER calcium depletion and recovery. The disturbed Ca2+ flux causes ER stress and increased BiP, and dysregulates synthesis of proband type I collagen at multiple steps. Collagen helical lysine hydroxylation is reduced, while telopeptide hydroxylation is increased, despite increased LH1 and decreased Ca2+-dependent FKBP65, respectively. Although PDI levels are maintained, procollagen chain assembly is delayed in proband cells. The resulting misfolded collagen is substantially retained in TRIC-B null cells, consistent with a 50-70% reduction in secreted collagen. Lower-stability forms of collagen that elude proteasomal degradation are not incorporated into extracellular matrix, which contains only normal stability collagen, resulting in matrix insufficiency. These data support a role for TRIC-B in intracellular Ca2+ homeostasis, and demonstrate that absence of TMEM38B causes OI by dysregulation of calcium flux kinetics in the ER, impacting multiple collagen-specific chaperones and modifying enzymes.

  View details for DOI 10.1371/journal.pgen.1006156

  View details for Web of Science ID 000381050100024

  View details for PubMedID 27441836

  View details for PubMedCentralID PMC4956114

• Reconstitution of a transmembrane protein, the voltage-gated ion channel, KvAP, into giant unilamellar vesicles for microscopy and patch clamp studies. Journal of visualized experiments : JoVE Garten, M., Aimon, S., Bassereau, P., Toombes, G. E. 2015: 52281

  Abstract

  Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods - electroformation and gel-assisted swelling - to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

  View details for DOI 10.3791/52281

  View details for PubMedID 25650630

• Methyl-branched lipids promote the membrane adsorption of alpha-synuclein by enhancing shallow lipid-packing defects PHYSICAL CHEMISTRY CHEMICAL PHYSICS Garten, M., Prevost, C., Cadart, C., Gautier, R., Bousset, L., Melki, R., Bassereau, P., Vanni, S. 2015; 17 (24): 15589-15597

  Abstract

  Alpha-synuclein (AS) is a synaptic protein that is directly involved in Parkinson's disease due to its tendency to form protein aggregates. Since AS aggregation can be dependent on the interactions between the protein and the cell plasma membrane, elucidating the membrane binding properties of AS is of crucial importance to establish the molecular basis of AS aggregation into toxic fibrils. Using a combination of in vitro reconstitution experiments based on Giant Unilamellar Vesicles (GUVs), confocal microscopy and all-atom molecular dynamics simulations, we have investigated the membrane binding properties of AS, with a focus on the relative contribution of hydrophobic versus electrostatic interactions. In contrast with previous observations, we did not observe any binding of AS to membranes containing the ganglioside GM1, even at relatively high GM1 content. AS, on the other hand, showed a stronger affinity for neutral flat membranes consisting of methyl-branched lipids. To rationalize these results, we used all-atom molecular dynamics simulations to investigate the influence of methyl-branched lipids on interfacial membrane properties. We found that methyl-branched lipids promote the membrane adsorption of AS by creating shallow lipid-packing defects to a larger extent than polyunsaturated and monounsaturated lipids. Our findings suggest that methyl-branched lipids may constitute a remarkably adhesive substrate for peripheral proteins that adsorb on membranes via hydrophobic insertions.

  View details for DOI 10.1039/c5cp00244c

  View details for Web of Science ID 000356056000011

  View details for PubMedID 25824255