The enigmatic nucleus of the marine dinoflagellateProrocentrum cordatum

Abstract

The marine, bloom-forming dinoflagellateProrocentrum 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-specificnucleus (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 flattenednucleus revealed highest density of nuclear pores in the vicinity of the nucleolus, a total of 62 tightly packed chromosomes (∼0.4-6.7 μm3), and interaction of several chromosomes with the nucleolus and other nuclear structures. A specificprocedure 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 identificationof 4,052 proteins (39% of unknown function), out of which 418 were predicted to serve specificnuclear 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 differingfrom those of typical eukaryotic cells.