Markus Gan­ter, Ph.D.

markus.ganter@med.uni-heidelberg.de

Phone: ++49-(0)6221–56-6546

Fax: ++49-(0)6221–56-5751

Nuclear Autonomy in Multinucleated Cells

Projects

Cells usu­al­ly mul­ti­ply by dupli­cat­ing their genome and divide them­selves into two daugh­ter cells. Yet, in some organ­isms, cel­lu­lar mul­ti­pli­ca­tion fol­lows dis­tinct, some­what bizarre routes. One of these rather unusu­al ways can be seen in Plas­mod­i­um spp., the causative agent of malar­ia. An unusu­al fea­ture of Plas­mod­i­umrepli­ca­tion inside ery­thro­cytes, known as schizo­gony, is the num­ber of nuclei and, hence, daugh­ter cells they pro­duce dur­ing one round of intra­cel­lu­lar repli­ca­tion. Par­a­sites with odd num­bers of nuclei can be read­i­ly seen (Fig. 1). Diver­gence from a geo­met­ric expan­sion (e. 2, 4, 8, 16, or 32) is caused by asyn­chro­nous repli­ca­tion, sug­gest­ing that indi­vid­ual nuclei repli­cate autonomous­ly until a glob­al mech­a­nism takes over and co-ordi­nates daugh­ter cell for­ma­tion. This is in strik­ing con­trast with nuclear divi­sion in oth­er mult­i­n­u­cle­at­ed cells, such as the ear­ly Drosophi­la embryo, where nuclei fol­low a geo­met­ric expan­sion, as their divi­sion is rel­a­tive­ly syn­chro­nous. To gain insight into the reg­u­la­tion of Plas­mod­i­um fal­ci­parum repli­ca­tion, we con­duct­ed a for­ward genet­ic screen for the con­di­tion­al pro­tein expres­sion of pro­tein kinas­es and iden­ti­fied a kinase as essen­tial for pro­lif­er­a­tion (Fig. 2). This kinase is Plas­mod­i­um-spe­cif­ic and a cru­cial reg­u­la­tor of the con­tin­u­ous rounds of DNA repli­ca­tion, his­tone mod­i­fi­ca­tion, and reg­u­la­tion of gene expres­sion. We also found that this kinase is required for trans­mis­sion to the mos­qui­to. This work serves as a start­ing point to gain a bet­ter under­stand­ing of nuclear auton­o­my in Plas­mod­i­um.

Fig­ure 1 | Nuclei divide autonomous­ly in Plas­mod­i­um. Odd num­bers of nuclei can be read­i­ly seen dur­ing the devel­op­ment of P. fal­ci­parum.

Fig­ure 2 | Plas­mod­i­um fal­ci­parum CRK4 is essen­tial for repli­ca­tion in the path­o­gen­ic blood stage of infec­tion.

1 | Analysis the spatiotemporal parameters of nuclear autonomy

To ana­lyze the spa­tiotem­po­ral para­me­ters of nuclear auton­o­my, we are using live-cell microscopy. In a set of reporter par­a­sites, we deter­mine the dynam­ics of DNA repli­ca­tion, nuclear divi­sion, and organelle devel­op­ment. These data inform on a math­e­mat­i­cal mod­el, describ­ing nuclear auton­o­my in P. fal­ci­parum (Fig. 3).

Fig­ure 3 | Plas­mod­i­um fal­ci­parum reporter cell line. The nuclei of the par­a­site are marked with a red-flu­o­res­cent pro­tein.

2 | Regulation of autonomous nuclei replication

To under­stand how autonomous nuclei reg­u­late their repli­ca­tion, we want to describe the sig­nalling cas­cade that orches­trates DNA repli­ca­tion in indi­vid­ual nuclei. For this we are using the may­or S‑phase reg­u­la­tor PfCRK4 as a start­ing point and are cur­rent­ly defin­ing its inter­ac­tome. This work will inform on sub­strates of the kinase as well as on upstream reg­u­la­tors.

Fig­ure 4 | Car­toon rep­re­sent­ing the elu­sive sig­nalling cas­cade, which trig­gers PfCRK4.

3 | Function of regulators and effectors in asynchromous DNA replication

Our pre­vi­ous work sug­gests that a set of pro­teins of unknown func­tion is involved in DNA repli­ca­tion and nuclear divi­sion. These pro­teins were iden­ti­fied through phos­pho­pro­teom­ic pro­fil­ing of PfCRK4 and showed a much-reduced phos­pho­ry­la­tion in absence of the kinase. To char­ac­ter­ize the func­tion of these reg­u­la­tors and effec­tors involved in asyn­chro­nous DNA repli­ca­tion, we are using reverse genet­ic tools and inducible gene-deple­tion approach­es.

Fig­ure 5 | Change in phos­pho­ry­la­tion of three pro­teins of unknown func­tion in absence of CRK4. Phos­pho­pro­teom­ic pro­fil­ing of PfCRK4-deplet­ed par­a­sites. Grey areas indi­cate phos­pho­pep­tides with a ≥2‑fold change in phos­pho­ry­la­tion and p<0.05.

4 | Collaboration with Zendia GmbH

In col­lab­o­ra­tion with the bio­med­ical start-up com­pa­ny Zen­dia GmbH and the Frischknecht lab at CIID, we are devel­op­ing a nov­el rapid diag­nos­tic test (RDT) to detect infec­tions with Plas­mod­i­um fal­ci­parum. Unlike oth­er RDTs, this sys­tem is able to enrich and detect par­a­sites from periph­er­al blood, allow­ing for an ear­ly onset of anti-malar­ia treat­ment.

Selected Publications

Com­plete pub­li­ca­tion list (PubMed)

Przy­bors­ki J. and Gan­ter M. (2018). Das ungewöhn­liche Leben der Malar­i­a­par­a­siten. Biol. Unser­er Zeit 48, 162–169 (invit­ed review arti­cle in Ger­man)

Gan­ter M., Gold­berg J.M., Dvorin J.D., Paulo J.A., King J.G., Tri­pathi A.K., Paul A.S., Yang J., Cop­pens I., Jiang R.H.Y., Elsworth B., Bak­er D.A., Din­glasan R.R., Gygi S.P., Durais­ingh M.T.  (2017). Plas­mod­i­um fal­ci­parum CRK4 directs con­tin­u­ous rounds of DNA repli­ca­tion dur­ing schizo­gony. Nat. Micro­bi­ol. 2(5):17017

Paul A.S., Saha S., Engel­berg K., Jiang R.H.Y., Cole­man B.I., Kos­ber A.L., Chen C., Gan­ter M., Espy N., Gilberg­er T.W., Gubbels M.J., Durais­ingh M.T. (2015). Par­a­site cal­cineurin reg­u­lates host cell recog­ni­tion and attach­ment by api­com­plex­ans. Cell Host Microbe. 18:49–60

Gan­ter M., Rizopou­los Z., Schuler H., Matuschews­ki K. (2015). Piv­otal and dis­tinct role for Plas­mod­i­um actin cap­ping pro­tein alpha dur­ing blood infec­tion of the Malar­ia par­a­site. Mol. Micro­bi­ol. 96:84–94

Cole­man B.I., Skill­man K.M., Jiang R.H.Y., Childs L.M., Altenhofen L.M., Gan­ter M., Leung Y., Goldowitz I., Kaf­sack B.F.C., Mar­ti M., Lli­nas M., Buc­k­ee C.O., Durais­ingh M.T. (2014). A Plas­mod­i­um fal­ci­parum his­tone deacety­lase reg­u­lates anti­genic vari­a­tion and game­to­cyte con­ver­sion. Cell Host Microbe. 16:177–186

*Sat­tler J., *Gan­ter M., Hliscs M., Matuschews­ki K., Schuler H. (2011). Actin reg­u­la­tion in the malar­ia par­a­site. Eur. J. Cell Biol. 90:966–971 (*equal con­tri­bu­tion)

*Siden-Kiamos I., *Gan­ter M., Kun­ze A., Hliscs M., Stein­büchel M., Men­doza J., Sin­den R., Louis K., Matuschews­ki K. (2011). Stage-spe­cif­ic deple­tion of myosin A sup­ports an essen­tial role in motil­i­ty of malar­i­al ookinetes. Cell Micro­bi­ol. 13(12):1996–2006 (*equal con­tri­bu­tion)

Gan­ter M., Schuler H., Matuschwes­ki K. (2009). Vital role for the Plas­mod­i­um cap­ping pro­tein (CP) beta sub­unit in motil­i­ty of malar­ia sporo­zoites. Mol. Micro­bi­ol. 74:1356–1367

Kur­su­la I., Kur­su­la P., Gan­ter M., Pan­jikar S., Matuschews­ki K., Schuler H. (2008). Struc­tur­al basis for par­a­site-spe­cif­ic func­tions of the diver­gent pro­fil­in of Plas­mod­i­um fal­ci­parum. Struc­ture. 16:1638–1648.