Kliniken & Institute … Institute Zentrum für… Parasitology Unit Research Current Groups Guizetti Lab

Malaria research: Guizetti

The Guizetti lab from left to right (May 2022): Caroline Simon (PhD student, alumna), Anja Klemmer (PhD student), Christoph Wenz (MSc student), Yannik Voß (PhD student), Nicolas Lichti (MSc student), Vanessa Stürmer (MSc student), Julien Guizetti (group leader)

We use advanced microscopy to study the proliferation of malaria-causing parasites in human blood

Lab members

Dr. Julien Guizetti (Group leader 2017-present)

Anja Klemmer (PhD student 2019-present)

Yannik Voß (MSc thesis 2018, PhD student 2019-present)

Nicolas Lichti (BSc thesis 2019, MSc thesis 2022-present)

Vanessa Stürmer (intern 2018-2019, MSc rotation 2020-2021, MSc thesis 2022-present)

Christoph Wenz (MSc rotation 2021, MSc thesis 2022-present)

Sophie Stopper (MSc rotation 2022-present)

Caroline Simon (PhD Student 2018-2022)

Ann-Kathrin Mehnert (MSc rotation 2018, MSc thesis 2019)

Marlen Wesemeyer (BSc thesis 2018)

Nadja Ilkenhans (BSc thesis 2018)

Johanna Bauer (BSc thesis 2020, MSc rotation 2021)

Nathan Ribot (MSc rotation 2020)

Dr. Tatiany Patricia Romão (Visiting Scientist, 2020-2021)

Marius Flörchinger (BSc thesis 2021, MSc rotation 2021)

We are always looking for motivated people to join our team and aim to promote scientific as well as personal development!

Figure 1. Schematics of the intra-erythrocytic development cycle of Plasmodium parasites. After invading a red blood cell and undergoing a growth phase the parasite first multiplies its nuclei (blue). Only at the end of the cycle the nuclei are packaged into daughter cell, which egress from the host cell to start a new cycle.

Research Interest

The severity of the malaria disease, which still causes the death of about 600’000 people per year, is predicted by the number of Plasmodium-infected red blood cells that circulate in the blood stream. Yet, we only have a rudimentary understanding about the unconventional cell division mechanism the parasite uses for its rapid proliferation. Contrary to most model organisms, which divide by binary fission, malaria-causing parasites first multiply their nuclei and later generate 8-30 daughter cells at once (Fig. 1). I

n the Guizetti group we study the mechanism of nuclear division to ultimately uncover how the parasite achieves its final number of daughter cells. We use modern super-resolution, electron, and live cell microscopy technologies combined with CRISPR/Cas9 genome editing to describe the kinetics of nuclear multiplication, study the organization and composition of the centrosome, and aim to uncover the putative role of phase separation in centrosome function (see projects). By studying this divergent eukaryote, we are contributing an evolutionary cell biology perspective, but also aim to uncover new targets within this essential pathway that can be leveraged in the fight against malaria.

  • In "einfachen" Worten:

    Malaria ist eine schwere Krankheit, an der immer noch mehr als ein halbe Million Menschen jährlich versterben. Die Krankheit entsteht während sich der Malaria-Erreger, genannt Plasmodium falciparum, im Blut eines infizierten Menschen massiv vermehrt. Nach einem infektiösen Mückenstich und einem Zwischenstopp in der Leber dringt der kleine Erreger in unsere roten Blutzellen ein. Dort verdaut er die Blutzelle von innen und wächst. In der zweiten Hälfte dieses Entwicklungszyklus beginnt er seine eigenen Zellkerne, die das Erbmaterial in Form von DNA enthalten, zu vermehren. Erst zum Schluss seiner Entwicklung verpackt er die Zellkerne in 8 bis 30 Tochterzellen, die dann aus der platzenden Wirtszelle entkommen, um dann viele neue rote Blutzellen zu infizieren. Dieser Vermehrungsprozess unterscheidet sich signifikant von dem, der sich in z.B. unseren Körperzellen abspielt, die üblicherweise eine Zweiteilung durchlaufen. Auch deshalb verstehen wir die Zellteilung des Parasiten recht wenig. Ziel der AG Guizetti ist es mithilfe von modernen Mikroskopietechniken diesen Prozess mit einer hohen zeitlichen und räumlichen Auflösung zu untersuchen und zu verstehen (siehe Filme und Bilder). Wir hoffen, damit neue Zielmoleküle für Interventionsstrategien im Kampf gegen Malaria aufzudecken.

    Mehr Infos zu Malaria gibt es in unserem Service Bereich!

Projects

Please click on the image to start the movie!

Cell division is a highly dynamic process. In the case of malaria parasites, key cellular parameters have, however, not been assessed in a time-resolved manner. We develop time-lapse imaging approaches combined with live cell staining protocols to determine division timing, nuclear multiplication rates, and spindle reorganization and more at the level of individual parasites (Fig. 2). We are thereby creating a quantitative cell biological framework that will enable the study of malaria parasite cell division with unprecedented spatial and temporal resolution.

Figure 2. Malaria-parasites produce multiple nuclei before egressing from host cell. Time lapse movie of parasite infected red blood cell from start of nuclear division until bursting and reinvasion of neighboring cell. Red blood cell and parasite can be seen in transmission channel (grey), while the chromatin, i.e. the nuclei, of the parasite are fluorescently labeled by expression of GFP-tagged Histone 2B (green).

Nuclear division requires small and intricate molecular machines that physically pull apart chromosomes after their replication. Key components of this machinery are i) the mitotic spindle, which consists of tube-shaped polymers called microtubules, to which individual chromosomes are attached and ii) the centrosomes which organizes the microtubules and form the two poles to which the chromosomes are pulled (Fig. 3). The organization of the surprisingly small mitotic spindle and the atypical centrosome in Plasmodium has not been well studied. Using a combination of super-resolution and electron microscopy we have created the first detailed working model of the malaria parasite centrosome (Fig. 3). We can now use this model to step-by-step identify the components of this divergent organelle and elucidate their function.

Figure 3. Working model of the malaria parasite centrosome and mitotic spindle. Microtubules (magenta) form a bipolar array called mitotic spindle. The spindle pole, also called centrosome (grey), is a protein dense structure that spans the nuclear membrane (grey blue) through a special nuclear pore (red). The extranuclear part contains centrins (green) one of the few conserved centrosomal protein families. The intranuclear part harbors microtubule nucleation site (white) from which microtubules extend towards the nuclear DNA (blue). Later this mitotic spindle will extend to drive apart the sister chromosomes. Adapted from Simon et al. 2021 (PMID: 34535568).

Please click on the image to start the movie!

Centrosomes can be viewed as membrane-less organelles that concentrate many different components in a defined sub-cellular compartment. In recent years the new biophysical concept of phase separation, which describes the propensity of certain proteins to coacervate under specific conditions, has been employed to explain the properties of membrane-less organelles including the centrosomes. Phase-separation has not yet been investigated in malaria-causing parasites, but when studying recombinant P. falciparum centrins, which are conserved calcium-binding proteins, in vitro we found that some can undergo liquid-liquid phase separation (Fig. 4). The importance of this feature for centrosome assembly and function in vivo is the subject of further studies in the group.

Figure 4. Recombinant centrin protein droplets display liquid-like behavior. Transmission time-lapse microscopy of a recombinant PfCen1 protein solution after addition of calcium. Fusion between two protein droplets indicates that they constitute liquid-liquid phase condensates rather than solid protein assemblies. Adapted from Voss et al. 2022 (biorxiv.org/content/10.1101/2022.07.26.501452v1).

Movie gallery

Movie 1 - Malaria parasites generates multiple daughter cells that invade neighboring erythrocytes.

Time lapse movie of parasite infected red blood cell which bursts and releases multiple daughter cells that invade neighboring red blood cells where they start the next developmental cycle. Red blood cells and parasites can be seen in transmission channel (grey), while parts of the chromosomes of the parasites is fluorescently by GFP-tagged HP1 (green) (kindly provided by Till Voss, Basel).

Movie 2 - Highly resolved three-dimensional organization of microtubules and centrosomes in multinucleated malaria parasite.

3D-rendered ultrastructure expansion microscopy image of Plasmodium falciparum blood stage schizont parasite with centrosomes and microtubules labeled using anti-centrin (green) and anti-tubulin (magenta), while nuclear DNA is stained with Hoechst (blue). Cells was isotropically expanded by a factor of 4.5.

Movie 3 - Multiple rounds mitotic spindle formation and extension malaria parasite.

Super-resolution time-lapse confocal microscopy of 3D7 wild type blood stage parasite labelled with microtubule live cell dye SPY555-Tubulin (magenta) and acquisition of transmission mode (grey) undergoing the first few mitotic divisions. Images were acquired with Zeiss Airyscan detector and processed for improved resolution. Maximum projections are shown. Image dimensions are 10 x 10 µm.

Movie 5 - Three-dimensional organization of microtubules, nuclear pores, and mitotic spindle in dividing nuclei using ultrastructure expansion microscopy.

3D-rendering of a deconvolved confocal z-stack (17x300nm) acquired of a hemispindle-containing nucleus in a Nup313-3xHA_glms expressing 3D7 schizont parasite labeled with anti-centrin (green), anti-tubulin (magenta), anti-HA (yellow) antibodies and stained with Hoechst (blue) after isotropic expansion by a factor of 4.5. Maximum Intensity Projection (MIP) mode in Imaris was used. Scale bars: 1 µm.

Movie 4 - Three-dimensional organization of microtubules, centriolar plaques, nuclear pores and hemispindle in dividing nuclei using ultrastructure expansion microscopy.

3D-rendering of a deconvolved confocal z-stack (17x300nm) acquired of a hemispindle-containing nucleus in a Nup313-3xHA_glms expressing 3D7 schizont parasite labeled with anti-centrin (green), anti-tubulin (magenta), anti-HA (yellow) antibodies and stained with Hoechst (blue) after isotropic expansion by a factor of 4.5. Maximum Intensity Projection (MIP) mode in Imaris was used. Scale bars: 1 µm.

Publications / Collaborations

Ganter M*, Guizetti J*, Kilian N* (2022) Visualization of Infected Red Blood Cell Surface Antigens by Fluorescence Microscopy In: Jensen ATR, Hviid L, editors. Malaria Immunology: Targeting the Surface of Infected Erythrocytes. New York, NY: Springer US. pp. 425–433. doi:10.1007/978-1-0716-2189-9_31

Voss Y, Klaus S, Ganter M, Guizetti J* (2022) Ca2+-inducible phase separation of centrins in proliferating malaria parasites. bioRxiv 2022.07.26.501452. doi:10.1101/2022.07.26.501452

Klaus S, Binder P, Kim J, Machado M, Funaya C, Schaaf V, Klaschka D, Kudulyte A, Cyrklaff M, Laketa V, Höfer T, Guizetti J, Becker NB, Frischknecht F, Schwarz US, Ganter M (2022) Asynchronous nuclear cycles in multinucleated Plasmodium falciparum facilitate rapid proliferation. Sci Adv. Apr;8(13):eabj5362. doi: 10.1126/sciadv.abj5362. Epub 2022 Mar 30. PMID: 35353560.

Machado M, Klaus S, Klaschka D, Guizetti J, Ganter M (2022) Plasmodium falciparum CRK4 links early mitotic events to the onset of S-phase during schizogony. bioRxiv 2022.08.31.505163. doi:10.1101/2022.08.31.505163.

Wenz C, Simon CS, Romão TP, Stürmer V, Machado M, Klages N, Klemmer A, Voß Y, Ganter M, Brochet M, Guizetti J* (2022) An Sfi1-like centrin-interacting centriolar plaque protein affects nuclear microtubule homeostasis. bioRxiv 2022.07.28.501831. doi:10.1101/2022.07.28.501831

Diehl M, Roling L, Rohland L, Weber S, Cyrklaff M, Sanchez CP, Beretta CA, Simon CS, Guizetti J, Hahn J, Schulz N, Mayer MP, Przyborski JM (2021) Co-chaperone involvement in knob biogenesis implicates host-derived chaperones in malaria virulence. PLoS Pathog. Oct 6;17(10):e1009969. doi: 10.1371/journal.ppat.1009969. PMID: 34614006; PMCID: PMC8544838.

Simon CS, Funaya C, Bauer J, Voβ Y, Machado M, Penning A, Klaschka D, Cyrklaff M, Kim J, Ganter M, Guizetti J (2021) An extended DNA-free intranuclear compartment organizes centrosome microtubules in malaria parasites. Life Sci Alliance. Sep 17;4(11):e202101199. doi: 10.26508/lsa.202101199. PMID: 34535568; PMCID: PMC8473725.

Simon CS, Stürmer VS, Guizetti J (2021) How Many Is Enough? - Challenges of Multinucleated Cell Division in Malaria Parasites. Front Cell Infect Microbiol. May 7;11:658616. doi: 10.3389/fcimb.2021.658616. PMID: 34026661; PMCID: PMC8137892.

Soni K, Kempf G, Manalastas-Cantos K, Hendricks A, Flemming D, Guizetti J, Simon B, Frischknecht F, Svergun DI, Wild K, Sinning I (2021) Structural analysis of the SRP Alu domain from Plasmodium falciparum reveals a non-canonical open conformation. Commun Biol. May 20;4(1):600. doi: 10.1038/s42003-021-02132-y. PMID: 34017052; PMCID: PMC8137916.

Guizetti J, Frischknecht F (2021) Apicomplexans: A conoid ring unites them all. PLoS Biol. Mar 11;19(3):e3001105. doi: 10.1371/journal.pbio.3001105. PMID: 33705378; PMCID: PMC7951970.

Barcons-Simon A, Cordon-Obras C, Guizetti J, Bryant JM, Scherf A. (2020) CRISPR Interference of a Clonally Variant GC-Rich Noncoding RNA Family Leads to General Repression of var Genes in Plasmodium falciparum. mBio. Jan 21;11(1)

Mehnert, A.K., Simon C.S., Guizetti, J. (2019). Immunofluorescence staining protocol for STED nanoscopy of Plasmodium-infected red blood cells. Mol Biochem Parasitol. 229, 47–52.

Müller, L. S. M., Cosentino, R. O., Förstner, K. U., Guizetti, J., Wedel, C., Kaplan, N., … Siegel, T. N. (2018). Genome organization and DNA accessibility control antigenic variation in trypanosomes. Nature 563(7729), 121–125

Zanghì, G., Vembar, S. S., Baumgarten, S., Ding, S., Guizetti, J., Bryant, J. M., … Scherf, A. (2018). A Specific PfEMP1 Is Expressed in P. falciparum Sporozoites and Plays a Role in Hepatocyte Infection. Cell Reports 22(11), 2809–2817.

Bryant J.M., Regnault C., Scheidig-Benatar C., Baumgarten S., Guizetti J.*, Scherf A. (2017). CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for var2csa Gene Activation or Silencing in Plasmodium falciparum. MBio 8(4). pii: e00729-17.

Guizetti, J.*, Barcons-Simon, A., Scherf, A. (2016). Trans-acting GC-rich non-coding RNA at var expression site modulates gene counting in malaria parasite. Nucleic Acids Res 44, 9710–9718.

Zhang, Q., Siegel, T. N., Martins, R. M., Wang, F., Cao, J., Gao, Q., Cheng, X., Jiang, L., Hon, C. C., Scheidig-Benatar, C., Sakamoto, H., Turner, L., Jensen, A. T., Claes, A., Guizetti, J., Malmquist, N. A., and Scherf, A. (2014). Exonuclease-mediated degradation of nascent RNA silences genes linked to severe malaria. Nature 513, 431–435.

Guizetti, J., Martins, R. M., Guadagnini, S., Claes, A., and Scherf, A. (2013). Nuclear Pores and Perinuclear Expression Sites of var and Ribosomal DNA Genes Correspond to Physically Distinct Regions in Plasmodium falciparum. Eukaryot Cell 12, 697–702.

Guizetti J*, Scherf A. (2013). Silence, activate, poise and switch! Mechanisms of antigenic variation in Plasmodium falciparum. Cell Microbiol. doi:10.1111/cmi.12115.

Guizetti J, Gerlich DW. (2012). ESCRT-III polymers in membrane neck constriction. Trends Cell Biol 22:133–140. doi:10.1016/j.tcb.2011.11.007.

Guizetti, J., Schermelleh, L., Mantler, J., Maar, S., Poser, I., Leonhardt, H., Muller-Reichert, T., and Gerlich, D. W. (2011). Cortical constriction during abscission involves helices of ESCRT-III-dependent filaments. Science 331, 1616–1620.

Guizetti, J., Mantler, J., Muller-Reichert, T., and Gerlich, D. W. (2010). Correlative time-lapse imaging and electron microscopy to study abscission in HeLa cells. Methods Cell Biol 96, 591–601.

Lacroix, B., van Dijk, J., Gold, N. D., Guizetti, J., Aldrian-Herrada, G., Rogowski, K., Gerlich, D. W., and Janke, C. (2010). Tubulin polyglutamylation stimulates spastin-mediated microtubule severing. J Cell Biol 189, 945–954.

Guizetti J, Gerlich DW. (2010). Cytokinetic abscission in animal cells. Semin Cell Dev Biol 21:909–916. doi:10.1016/j.semcdb.2010.08.001.

Guizetti J, Mäntler J, Müller-Reichert T, Gerlich DW. (2010). Correlative time-lapse imaging and electron microscopy to study abscission in hela cells. Methods Cell Biol 96:591–601. doi:10.1016/S0091-679X(10)96024-X.

Lekomtsev, S., Guizetti, J., Pozniakovsky, A., Gerlich, D. W., and Petronczki, M. (2010). Evidence that the tumor-suppressor protein BRCA2 does not regulate cytokinesis in human cells. J Cell Sci 123, 1395–1400.

Steigemann, P., Wurzenberger, C., Schmitz, M. H., Held, M., Guizetti, J., Maar, S., and Gerlich, D. W. (2009). Aurora B‑mediated abscission checkpoint protects against tetraploidization. Cell 136, 473–484.

Guizetti, J.*, and Scherf, A. (2013). Silence, activate, poise and switch! Mechanisms of antigenic variation in Plasmodium falciparum. Cell Microbiol 15, 718–726.

Guizetti, J., and Gerlich, D. W. (2012). ESCRT-III polymers in membrane neck constriction. Trends Cell Biol 22, 133–140.

Guizetti, J., and Gerlich, D. W. (2010). Cytokinetic abscission in animal cells. Semin Cell Dev Biol 21, 909–916.

List of preprints complete list of publications - Pubmed

Group activities

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Defense party - Caroline Simon (16 May 2022)

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Proud new Dr. rer. nat. Caroline Simon

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Trip to the zoo II (2022)

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Virtual X-mas party (2021)

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Trip to the zoo I (2021)

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Lab excursion (2021)

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Socially distanced lab meeting (2020)

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STED easter egg (2020)

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Merry X-mas group picture (2019)

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X-mas dinner impression I (2019)

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X-mas dinner impression II (2019)

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X-mas presents (2019)

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"Nacht der Forschung" (27 September 2019)

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"Bad taste" party (2019)

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Lab excursion (2018)

News - Guizetti group

New paper in "Life Science Alliance"

(publication date: 17 September 2021)

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In the new article the Guizetti lab and colleagues provide a first model of the atypical parasite centrosome, which is essential for orchestrating the rapid cell division of the Plasmodium parasite causing malaria:

An extended DNA-free intranuclear compartment organizes centrosome microtubules in malaria parasites.  Simon CS, Funaya C, Bauer J, Voss Y, Machado M, Penning A, Klaschka D, Cyrklaff M, Kim J, Ganter M, Guizetti J. 2021. Life Science Alliance doi:10.26508/lsa.202101199

Seminar series "Moment for Research"

Online-lecture by our group leader Julien

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As part of the Daimler and Benz Foundation's monthly seminar series "Moment for Research", our group leader Julien reports on his research in a 15-minute online lecture entitled "Malaria Pathogens in the Blood – Multiplying and Hiding" (Please note: the lecture is in German). The series offers insights into current research topics funded by the Daimler and Benz Foundation. The protagonists are scholarship holders of the postdoctoral funding programme who present their individual projects. So there is something exciting to discover every month!

How to get in touch:

Dr. Julien Guizetti

Centre for Infectious Diseases, Parasitology
Heidelberg University Hospital 
Im Neuenheimer Feld 324 (office & labs in INF 344, 2nd floor)
69120 Heidelberg, Germany

Tel. +49 6221 56-7877 (office) / -7882 (lab)

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