Knockdown of Dinoflagellate Condensin CcSMC4 Subunit Leads to S-Phase Impediment and Decompaction of Liquid Crystalline Chromosomes

Abstract

Dinoflagellates have some of the largest genomes, and their liquid-crystalline chromosomes (LCCs) have high degrees of non-nucleosomal superhelicity with cation-mediated DNA condensation. It is currently unknown if condensins, pentameric protein complexes containing structural maintenance of chromosomes 2/4, commonly involved in eukaryotic chromosomes condensation in preparation for M phase, may be involved in the LCC structure. We find that CcSMC4p (dinoflagellate SMC4 homolog) level peaked at S/G2 phase, even though LCCs do not undergo global-decondensation for replication. Despite the differences in the chromosomal packaging system, heterologous CcSMC4p expression suppressed conditional lethality of the corresponding fission yeast mutant, suggesting conservation of some canonical condensin functions. CcSMC4p-knockdown led to sustained expression of the S-phase marker PCNAp, S-phase impediment, and distorted nuclei in the early stage of CcSMC4p depletion. Prolonged CcSMC4p-knockdown resulted in aneuploidal cells and nuclear swelling with increasing LCC decompaction–decondensation. Cumulatively, our data suggested CcSMC4p function was required for dinoflagellate S-phase progression, and we propose that condensin-mediated higher-order compaction provisioning is involved in the provision of local rigidity for the replisome.

Dinoflagellate Phosphopantetheinyl Transferase (PPTase) and Thiolation Domain Interactions Characterized Using a Modified Indigoidine Synthesizing Reporter

“…Protists, in particular, representing a huge amount of eukaryotic evolutionary diversity, have lagged behind, although new sequencing technologies have provided a much-needed boost in investigative power [53], resulting in several partial genomes for dinoflagellates in particular [54][55][56][57][58]. Some genetic modifications of dinoflagellates have been successful for the purpose of harnessing the unique lipids of marine protists [59] focusing on knockdowns and knockouts [60,61] but also to investigate the unique biology of dinoflagellates [62]. Success using these techniques is generally limited due to the high copy number of many dinoflagellate genes [18], especially for precision techniques such as CRISPR/Cas9.…”

Oleaginous Heterotrophic Dinoflagellates—Crypthecodiniaceae

“…likely contained independent species [ 11 ], but no further characterization was conducted. This taxonomic position is thus counterintuitive, given its industrial and cell biology significance, with the organisms being the only colony-forming dinoflagellates with published mutants [ 27 , 28 , 29 , 30 , 31 , 32 , 33 ], and given the availability of the inducible strain crossing method [ 33 ], a true minimal medium without the use of undefined seawater [ 34 ], the cell cycle synchronization method, as well as efficient transformation protocols [ 29 , 35 , 36 , 37 , 38 ]. Relatively high oil content is an apparent genus trait, as all the strains in our laboratory collection exhibited at least one order of magnitude higher Nile red intensity than other dinoflagellates, indicating the group’s potential for biofuel production.…”

A Strategy for Gene Knockdown in Dinoflagellates

“…In particular, we are pursuing the use of an antisense-based knockdown approach in order to study how a decrease in target gene expression effects dinoflagellate metabolism. In prior research, the introduction of antisense-oligomers to dinoflagellate cells has been hampered by their thick, cellulosic cell wall [42,43]. Other studies have bypassed this obstacle by preparing spheroplasts, cells with a completely or partially removed cellwall, beforehand [42][43][44].…”

“…In prior research, the introduction of antisense-oligomers to dinoflagellate cells has been hampered by their thick, cellulosic cell wall [42,43]. Other studies have bypassed this obstacle by preparing spheroplasts, cells with a completely or partially removed cellwall, beforehand [42][43][44]. Spheroplast production is done by incubating cells on plates in a polyethylene glycol (PEG) solution, which promotes fusion of the vesicles and cell membrane, and ultimately a decrease in total cellulose.…”

“…Spheroplast production is done by incubating cells on plates in a polyethylene glycol (PEG) solution, which promotes fusion of the vesicles and cell membrane, and ultimately a decrease in total cellulose. So far only two studies have been successful in achieving gene knockdown with this spheroplast procedure; targeting a condensin subunit and targeting a cellulose synthase [42][43][44]. Once introduced, the antisense-oligo was able to bind to cytoplasmic mRNA and knockdown expression of the target gene.…”