INTRACELLULAR DYNAMICS AND NANOSCOPY
INTRACELLULAR DYNAMICS AND NANOSCOPY GROUP
Independent Senior Research Fellow
Intracellular Dynamics and Nanoscopy Group,
ICFO–Institute of Photonic Sciences,
The Barcelona Institute of Science and Technology
During my career, I have gained multidisciplinary expertise in theoretical biophysics, molecular cell biology and biochemistry, and advanced microscopy tools. This has allowed me to quantitatively tackle different
fundamental problems in cell biology, such as the mechanisms of membrane curvature generation and organelle homeostasis. During my PhD in theoretical biophysics (University of Barcelona and Tel Aviv University), I developed different theoretical and computational methods to study membrane dynamics and morphology. During my postdoc at CRG–Center for Genomic Regulation (Barcelona) I mastered a broad variety of biochemistry and molecular cell biology tools, with which I discovered novel ways of how lipids and proteins cooperate to functionally organize the Golgi membranes.
Currently, I lead the Intracellular Dynamics and Nanoscopy group at ICFO (Castelldefeles, Barcelona), an interdisciplinary group under the the umbrella of the Single Molecule Biophotonics Lab at ICFO, studying organelle morphology and dynamics, with a special focus on intracellular membrane fission. We combine advanced microscopy techniques (single-molecule fluorescence and superresolution nanoscopy), molecular and cell biology tools, with theoretical biophysics approaches to tackle highly controversial or still mysterious fundamental topics in cell biology with a clear pathophysiological relevance.
We are an interdisciplinary group working under the umbrella of the Single Molecule Biophotonics Lab at ICFO, studying organelle morphology and dynamics, with a special focus on intracellular membrane fission. We combine theoretical biophysics, molecular cell biology and advanced microscopy techniques to tackle highly controversial or still mysterious fundamental topics in cell biology with a clear pathophysiological relevance.
MORPHOLOGY AND DYNAMICS
Orderly organization and timing of Golgi membrane fission
The Golgi complex is the central organelle of the secretory pathway, where secretory proteins are sequentially glycosylated along the different cisternae, after which fully glycosylated proteins are exported in membrane-bound transport carriers for secretion. Sequential glycosylation is guaranteed by the polarized distribution of the enzymes along the Golgi stack. Thus, early acting glycosylation enzymes are concentrated in the cis cisternae, whereas late acting enzymes reside in the trans cisternae. What is the mechanism that ensures that only fully processed proteins are secreted? It seems that the Golgi membranes need to be laterally compartmentalized into regions that present the optimal microenvironment to efficiently promote these two different functions of the Golgi complex in a tightly regulated and hierarchical manner. In particular, export domains set the grounds for transport carrier formation (budding and fission), whereas protein processing occurs in enzymatic domains. However, due to their intrinsic dynamic character and small size (intra-Golgi vesicles have a uniform size of 60- 90 nm, and enzymatic domains are probably even smaller), experimental accessibility to such functional membrane domains has been challenging. A long-standing question in the field is how cargoes progress along the Golgi stack during protein transport (at export domains), while they are processed by Golgi-resident enzymes (at enzymatic domains). This problem can be split into two separate albeit interdependent questions: (i) what are the mechanisms of intra-Golgi membrane traffic? And (ii) how do Golgi-resident proteins separate from secretory cargoes after they functionally interact?
MEMBRANE MECHANICS AT THE ENDOPLASMIC RETICULUM
Mechanosensing and morphosensing in the regulation of membrane fission at the ER
Mechanosensation and morphosensation relate to the responsiveness of cellular signal transduction events to mechanical or geometrical cues, respectively. On one hand, integrin activation and focal adhesion formation at the plasma membrane are well-characterized examples of how mechanical perturbations can affect protein conformation and downstream signaling13. On the other hand, curvature sensing proteins are a good example of morphosensors, since their membrane binding affinity is dependent on a geometrical cue (in this case, membrane curvature). In the process of membrane fission, the size of the transport carriers –and therefore the timing of the fission event– normally responds to the structure of the proteins curving the membrane. However, in some special cases, the size of the transport carriers needs to be readjusted to commensurate with the size of the proteins therein contained. To study this fascinating process, we are focusing on how the ER budding machinery is modulated to generate transport carriers commensurate with the size of large cargos, such as collagens, a process dependent on a protein named TANGO1. We combine molecular cell biology and biochemistry, super-resolution nanoscopy, and quantitative physical modeling, and found that TANGO1, by assembling into a ring at ER exit sites, generates a semistable sub-domain across two compartments. The processes that allow this assembly also coordinately select, partition, and organize folded cargo, export machinery, and membrane acquisition for the generation of a transport carrier commensurate with the size of large cargos, such as collagens.
MECHANICAL AND TOPOLOGICAL PRINCIPLES OF MITOCHONDRIAL FISSION
Double membrane fission: the case of mitochondrial division
Mitochondria are double membrane-bound organelles, which are continuously undergoing fusion and fission, events crucial for cell physiology. Despite its importance, almost nothing is known about the energetic and topological requirements for double membrane fission. We will exploit our expertise in theoretical membrane biophysics to develop the first continuum elastic model of a double membrane to unveil the physical mechanisms of double membrane fission. We will implement both the lateral stress profile of the involved lipids and the protein-induced elastic stresses and strains (by protein wedging, membrane constriction, and inter-bilayer interactions).
We are a multidisciplinary group, which combines super-resolution nanoscopy and single molecule imaging techniques, molecular and cell biological tools, with theoretical biophysics to study organelle function.
MOLECULAR AND CELL BIOLOGY TOOLS
Quantitative super-resolution light nanoscopy and single molecule fluorescence techniques
To study the mechanisms driving the morphology and dynamics of intracellular organelles, we are applying a wide palette of molecular and cell biology tools, such as CRISPR/Cas9-mediated genome editing, cargo synchronization methods (RUSH system, FM4 aggregation domains), diverse protein dimerization assays (FKBP-FRB trapping assays, optogenetic CRY2/CIBN tools, bifluorescence complementation assays). We are also developing minimal system for in vitro reconstitution of cellular functions.
Physical modeling of membrane mechanics and cellular behavior
We are developing continuous elastic models of cellular membranes, organelle morphology and intracellular trafficking. We base our models on classical concepts of elasticity theory, statistical mechanics, and soft matter physics.
ADVANCED MICROSCOPY TECHNIQUES
Quantitative super-resolution light nanoscopy and single molecule fluorescence techniques
To characterize and monitor the organization, functionality and dynamics of intracellular organelles, such as the Golgi complex or the endoplasmic reticulum, we use cutting-edge imaging techniques, including super-resolution nanoscopy (STED and STORM) and single molecule techniques (intracellular single particle tracking), to overcome the spatial and temporal limitations of conventional diffraction-limited light microscopy techniques.
I. RAOTE, M. F. GARCIA-PARAJO, V. MALHOTRA, AND F. CAMPELO.
bioRxiv (2019). doi: 10.1101/578427
Y. WAKANA, K. HAYASHI, T. NEMOTO, C. WATANABE, M. TAOKA, F. CAMPELO, H. KUMATA, T. UMEMURA, H. INOUE, K. ARASAKI, M. TAGAYA.
bioRxiv (2019). doi: 10.1101/679936
J. COLLADO, M. KALEMANOV*, F. CAMPELO*, C. BOURGOINT*, F. THOMAS, R. LOEWITH, A. MARTINEZ-SÁNCHEZ, W. BAUMEISTER, C. J. STEFAN, AND R. FERNÁNDEZ-BUSNADIEGO.
Dev. Cell 51, 4 (2019), 476–487. doi: 10.1016/j.devcel.2019.10.018
L. BASSAGANYAS, S. J. POPA, M. HORLBECK, C. PURI, S. E. STEWART , F. CAMPELO, A. ASHOK, C. M. BUTNARU, N. BROUWERS, K. HEYDARI, J. RIPOCHE, J. WEISSMAN, D. C. RUBINSZTEIN, R. SCHEKMAN, V. MALHOTRA, K. MOREAU, AND J. VILLENEUVE.
New factors for protein transport identified by a genome-wide CRISPRi screen in mammalian cells.
J. Cell Biol. 218, 11 (2019), 3861–3879. doi: 10.1083/jcb.201902028
E. T. GARBACIK*, M. SANZ-PAZ*, K. J. E. BORGMAN, F. CAMPELO, AND M. F. GARCIA-PARAJO.
Frequency-encoded multicolor fluorescence imaging with single-photon counting color-blind detection.
Biophys. J., (2018). doi: 10.1016/j.bpj.2018.07.008
I. RAOTE, M. ORTEGA-BELLIDO, A. J. M. SANTOS, O. FORESTI, C. ZHANG, M. F. GARCIA-PARAJO, F. CAMPELO, AND V. MALHOTRA.
eLife 7, (2018), e32723. doi: 10.7554/eLife.32723
F. CAMPELO*, J. VAN GALEN, G. TURACCHIO, S. PARASHURAMAN, M. M. KOZLOV, M. F. GARCIA-PARAJO, AND V. MALHOTRA*.
Sphingomyelin metabolism controls the shape and function of the Golgi cisternae.
eLife 6, (2017), e24603. doi: 10.7554/eLife.24603
S. CAPASSO, L. STICCO, R. RIZZO, M. PIROZZI, D. RUSSO, N. A. DATHAN, F. CAMPELO, J. VAN GALEN, M. HÖLTTÄ-VUORI, G. TURACCHIO, A. HAUSSER, V. MALHOTRA, I. RIEZMAN, H. RIEZMAN, E. IKONEN, C. LUBERTO, S. PARASHURAMAN, A. LUINI, AND G. D’ANGELO.
Sphingolipid metabolic flow controls phosphoinositide turnover at the trans-Golgi network.
EMBO J. 36,12 (2017), 1736–1754. doi: 10.15252/embj.201696048
J. VAN GALEN*, F. CAMPELO*, E. MARTÍNEZ-ALONSO, M. SCARPA, J. Á. MARTÍNEZ-MENÁRGUEZ, AND V. MALHOTRA.
Sphingomyelin homeostasis is required to form functional enzymatic domains at the trans-Golgi network.
J. Cell Biol. 206, 5 (2014), 609–618. doi: 10.1083/jcb.201405009
M. M. KOZLOV, F. CAMPELO, N. LISKA, L. V. CHERNOMORDIK, S. J. MARRINK, AND H. T. MCMAHON.
Mechanisms shaping cell membranes.
Curr. Opin. Cell Biol. 29 (2014), 53–60. doi: 10.1016/j.ceb.2014.03.006
F. CAMPELO, C. ARNAREZ, S. J. MARRINK, AND M. M. KOZLOV.
Helfrich model of membrane bending: from Gibbs theory of liquid interfaces to membranes as thick anisotropic elastic layers.
Adv. Colloid Interface Sci. 208 (2014), 25–33. doi: 10.1016/j.cis.2014.01.018
F. CAMPELO AND M. M. KOZLOV.
Sensing membrane stresses by protein insertions.
PloS Comput. Biol. 10, 4 (2014), e1003556. doi: 10.1371/journal.pcbi.1003556
J. M. DURAN*, F. CAMPELO*, J. VAN GALEN*, T. SACHSENHEIMER, J. SOT, M. V. EGOROV, C. RENTERO, C. ENRICH, R. S. POLISHCHUK, F. M. GOÑI, B. BRÜGGER, F. WIELAND, AND V. MALHOTRA.
Sphigomyelin organization is required for vesicle biogenesis at the Golgi complex.
EMBO J. 31 (2012), 4535–4546. doi: 10.1038/emboj.2012.317
F. CAMPELO, A. CRUZ, J. PÉREZ-GIL, L. VÁZQUEZ, AND A. HERNÁNDEZ-MACHADO. Phase-field model for the morphology of monolayer lipid domains.
Eur. Phys. J. E. 35 (2012), 49. doi: 10.1140/epje/i2012-12049-2
F. CAMPELO AND V. MALHOTRA.
Membrane fission: the biogenesis of transport carriers.
Annu. Rev. Biochem. 81 (2012), 407–27. doi: 10.1146/annurev-biochem-051710-094912
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ICFO–Insitute of Photonic Sciences
Av. Carl Friedrich Gauss, 3
08860 Castelldefels (Barcelona)
+ 34 93 554 2225