TY - JOUR

T1 - The enigmatic nucleus of the marine dinoflagellate Prorocentrum cordatum

AU - Kalvelage, Jana

AU - Wöhlbrand, Lars

AU - Schoon, Robin-Alexander

AU - Zink, Fiona-Marine

AU - Correll, Christina

AU - Senkler, Jennifer

AU - Eubel, Holger

AU - Hoppenrath, Mona

AU - Rhiel, Erhard

AU - Braun, Hans-Peter

AU - Winklhofer, Michael

AU - Klingl, Andreas

AU - Rabus, Ralf

N1 - ACKNOWLEDGMENTS This study was supported by the Deutsche Forschungsgemeinschaft (DFG) within the framework of the Collaborative Research Center Roseobacter (SFB TRR 51) to R.R. and INST 86/1852-1 to A.K.. We are grateful to Thomas Heimerl (Marburg) for expert advice on AMIRA software; Jennifer Grünert (Munich), Edith Kieselhorst (service unit electron and light microscopy, Oldenburg), Silke Ammermann (Oldenburg), and Franziska Iwan (Wilhelmshaven) for technical assistance in sample preparation for electron microscopy; Petra Bolte (core facility fluorescence microscopy, Oldenburg) for expert advice in confocal microscopy; Julian Mannhaupt and Colin Zimmermann (both Oldenburg) for analysis of electron microscopic images; Christina Passmann and Petra Wagler (both Oldenburg) for assistance in photography and image processing; and Matthias Schröder (Oldenburg) for large dataset management and organization. R.R. conceived the study; J.K. conducted the cultivation and sampling; J.K. and M.H. conducted the light microscopy; J.K., M.H., and E.R. conducted the SEM and TEM; J.K., M.H., C.C., and A.K. conducted the FIB/SEM; J.K., R.A.S., and F.M.Z. performed the 3D reconstruction of nucleus; J.K., L.W., and E.R. developed the procedure for nuclei enrichment; J.K., L.W., J.S., H.E., and H.P.B. conducted the proteomic analyses; J.K. and L.W. managed the large proteomic dataset and conducted the functional categorization; J.K., M.W., and R.R. reconstructed the nuclear processes; J.K. and R.R. wrote the manuscript with contributions from L.W., M.W., A.K., H.P.B., E.R., and M.H. All authors agreed to the final version of the manuscript.

PY - 2023/8/24

Y1 - 2023/8/24

N2 - The marine, bloom-forming dinoflagellate Prorocentrum cordatum CCMP 1329 (formerly P. minimum) has a genome atypical of eukaryotes, with a large size of ~4.15 Gbp, organized in plentiful, highly condensed chromosomes and packed in a dinoflagellate-specific nucleus (dinokaryon). Here, we apply microscopic and proteogenomic approaches to obtain new insights into this enigmatic nucleus of axenic P. cordatum. High-resolution focused ion beam/scanning electron microscopy analysis of the flattened nucleus revealed highest density of nuclear pores in the vicinity of the nucleolus, a total of 62 tightly packed chromosomes (~0.4-6.7 µm 3), and interaction of several chromosomes with the nucleolus and other nuclear structures. A specific procedure for enriching intact nuclei was developed to enable proteomic analyses of soluble and membrane protein-enriched fractions. These were analyzed with geLC and shotgun approaches employing ion-trap and timsTOF (trapped-ion-mobility-spectrometry time-of-flight) mass spectrometers, respectively. This allowed identification of 4,052 proteins (39% of unknown function), out of which 418 were predicted to serve specific nuclear functions; additional 531 proteins of unknown function could be allocated to the nucleus. Compaction of DNA despite very low histone abundance could be accomplished by highly abundant major basic nuclear proteins (HCc2-like). Several nuclear processes including DNA replication/repair and RNA processing/splicing can be fairly well explained on the proteogenomic level. By contrast, transcription and composition of the nuclear pore complex remain largely elusive. One may speculate that the large group of potential nuclear proteins with currently unknown functions may serve yet to be explored functions in nuclear processes differing from those of typical eukaryotic cells. IMPORTANCE Dinoflagellates form a highly diverse group of unicellular microalgae. They provide keystone species for the marine ecosystem and stand out among others by their very large, unusually organized genomes embedded in the nuclei markedly different from other eukaryotic cells. Functional insights into nuclear and other cell biological structures and processes of dinoflagellates have long been hampered by the paucity of available genomic sequences. The here studied cosmopolitan P. cordatum belongs to the harmful algal bloom-forming, marine dinoflagellates and has a recently de novo assembled genome. We present a detailed 3D reconstruction of the P. cordatum nucleus together with comprehensive proteogenomic insights into the protein equipment mastering the broad spectrum of nuclear processes. This study significantly advances our understanding of mechanisms and evolution of the conspicuous dinoflagellate cell biology.

AB - The marine, bloom-forming dinoflagellate Prorocentrum cordatum CCMP 1329 (formerly P. minimum) has a genome atypical of eukaryotes, with a large size of ~4.15 Gbp, organized in plentiful, highly condensed chromosomes and packed in a dinoflagellate-specific nucleus (dinokaryon). Here, we apply microscopic and proteogenomic approaches to obtain new insights into this enigmatic nucleus of axenic P. cordatum. High-resolution focused ion beam/scanning electron microscopy analysis of the flattened nucleus revealed highest density of nuclear pores in the vicinity of the nucleolus, a total of 62 tightly packed chromosomes (~0.4-6.7 µm 3), and interaction of several chromosomes with the nucleolus and other nuclear structures. A specific procedure for enriching intact nuclei was developed to enable proteomic analyses of soluble and membrane protein-enriched fractions. These were analyzed with geLC and shotgun approaches employing ion-trap and timsTOF (trapped-ion-mobility-spectrometry time-of-flight) mass spectrometers, respectively. This allowed identification of 4,052 proteins (39% of unknown function), out of which 418 were predicted to serve specific nuclear functions; additional 531 proteins of unknown function could be allocated to the nucleus. Compaction of DNA despite very low histone abundance could be accomplished by highly abundant major basic nuclear proteins (HCc2-like). Several nuclear processes including DNA replication/repair and RNA processing/splicing can be fairly well explained on the proteogenomic level. By contrast, transcription and composition of the nuclear pore complex remain largely elusive. One may speculate that the large group of potential nuclear proteins with currently unknown functions may serve yet to be explored functions in nuclear processes differing from those of typical eukaryotic cells. IMPORTANCE Dinoflagellates form a highly diverse group of unicellular microalgae. They provide keystone species for the marine ecosystem and stand out among others by their very large, unusually organized genomes embedded in the nuclei markedly different from other eukaryotic cells. Functional insights into nuclear and other cell biological structures and processes of dinoflagellates have long been hampered by the paucity of available genomic sequences. The here studied cosmopolitan P. cordatum belongs to the harmful algal bloom-forming, marine dinoflagellates and has a recently de novo assembled genome. We present a detailed 3D reconstruction of the P. cordatum nucleus together with comprehensive proteogenomic insights into the protein equipment mastering the broad spectrum of nuclear processes. This study significantly advances our understanding of mechanisms and evolution of the conspicuous dinoflagellate cell biology.

UR - http://www.scopus.com/inward/record.url?scp=85168798085&partnerID=8YFLogxK

U2 - 10.1128/msphere.00038-23

DO - 10.1128/msphere.00038-23

M3 - Article

C2 - 37358287

VL - 8

SP - e0003823

JO - MSPHERE

JF - MSPHERE

SN - 2379-5042

IS - 4

ER -