Megan M. Aoki

The pre-mRNA processing factor 4 kinase (PRP4K) is an essential gene in animal cells, making interrogation of its function challenging. Here, we report the first knockout model for PRP4K in the social amoeba Dictyostelium discoideum, revealing a new function in splicing events controlling autophagy. When prp4k knockout amoebae underwent multicellular development, we observed defects in differentiation linked to abnormal autophagy and aberrant secretion of stalk cell inducer c-di-GMP. Autophagosome-lysosome fusion was found to be impaired after PRP4K loss in both human cell lines and amoebae. Mechanistically, PRP4K loss results in mis-splicing and reduced expression of the ESCRT-III gene CHMP4B in human cells and its ortholog vps32 in Dictyostelium, and re-expression of CHMP4B or Vps32 cDNA (respectively) restored normal autophagosome-lysosome fusion in PRP4K-deficient cells. Thus, our work reveals a novel PRP4K-CHMP4B/vps32 splicing circuit regulating autophagy that is conserved over at least 600 million years of evolution.

Cytokinins (CKs) are a class of growth‐promoting signaling molecules that affect multiple cellular and developmental processes. These phytohormones are well studied in plants, but their presence continues to be uncovered in organisms spanning all kingdoms, which poses new questions about their roles and functions outside of plant systems. Cytokinin production can be initiated by one of two different biosynthetic enzymes, adenylate isopentenyltransfases (IPTs) or tRNA isopentenyltransferases (tRNA‐IPTs). In this study, the social amoeba, Dictyostelium discoideum , was used to study the role of CKs by generating deletion and overexpression strains of its single adenylate‐IPT gene, iptA . The life cycle of D. discoideum is unique and possesses both single‐ and multicellular stages. Vegetative amoebae grow and divide while food resources are plentiful, and multicellular development is initiated upon starvation, which includes distinct life cycle stages. CKs are produced in D. discoideum throughout its life cycle and their functions have been well studied during the later stages of multicellular development of D. discoideum . To investigate potential expanded roles of CKs, this study focused on vegetative growth and early developmental stages. We found that iptA ‐deficiency results in cytokinesis defects, and both iptA ‐deficiency and overexpression results in dysregulated tricarboxylic acid (TCA) cycle and amino acid metabolism, as well as increased levels of adenosine monophosphate (AMP). Collectively, these findings extend our understanding of CK function in amoebae, indicating that iptA loss and overexpression alter biological processes during vegetative growth that are distinct from those reported during later development.

Cytokinins (CKs) are a diverse group of evolutionarily significant growth-regulating molecules. While the CK biosynthesis and signal transduction pathways are the most well-understood in plant systems, these molecules have been identified in all kingdoms of life. This review follows the recent discovery of an expanded CK profile in the social amoeba, Dictyostelium discoideum. A comprehensive review on the present knowledge of CK biosynthesis, signal transduction, and CK-small molecule interactions within members of Dictyostelia will be summarized. In doing so, the utility of social amoebae will be highlighted as a model system for studying the evolution of these hormone-like signaling agents, which will set the stage for future research in this area.

Cytokinins (CKs) are a family of evolutionarily conserved growth regulating hormones. While CKs are well-characterized in plant systems, these N⁶-substituted adenine derivatives are found in a variety of organisms beyond plants, including bacteria, fungi, mammals, and the social amoeba, Dictyostelium discoideum. Within Dictyostelium, CKs have only been studied in the late developmental stages of the life cycle, where they promote spore encapsulation and dormancy. In this study, we used ultra high-performance liquid chromatography-positive electrospray ionization-high resolution tandem mass spectrometry (UHPLC-(ESI+)-HRMS/MS) to profile CKs during the Dictyostelium life cycle: growth, aggregation, mound, slug, fruiting body, and germination. Comprehensive profiling revealed that Dictyostelium produces 6 CK forms (cis-Zeatin (cZ), discadenine (DA), N⁶-isopentenyladenine (iP), N⁶-isopentenyladenine-9-riboside (iPR), N⁶-isopentenyladenine-9-riboside-5′ phosphate (iPRP), and 2-methylthio-N⁶-isopentenyladenine (2MeSiP)) in varying abundance across the sampled life cycle stages, thus laying the foundation for the CK biosynthesis pathway to be defined in this organism. Interestingly, iP-type CKs were the most dominant CK analytes detected during growth and aggregation. Exogenous treatment of AX3 cells with various CK types revealed that iP was the only CK to promote the proliferation of cells in culture. In support of previous studies, metabolomics data revealed that DA is one of the most significantly upregulated small molecules during Dictyostelium development, and our data indicates that total CK levels are highest during germination. While much remains to be explored in Dictyostelium, this research offers new insight into the nature of CK biosynthesis, secretion, and function during Dictyostelium growth, development, and spore germination.

Cytokinins (CKs) are adenine derivatives that act as phytohormones. These signaling molecules control plant cell division and differentiation, organ growth and senescence, and orchestrate plant interactions with biotic and abiotic environments. While CKs are predominately recognized as plant-based substances, CKs have been found across different domains of life, including microorganisms, insects, mammals, and humans. In plants, CKs act at trace, often low femtomolar concentrations; therefore, sensitive and precise analytical techniques are required to accurately detect and quantify them from complex biological matrices. Here, we report the first comprehensive CK quantification method using a QExactive Orbitrap mass spectrometer in high-resolution with a parallel reaction monitoring (PRM) based approach. The current method progresses upon multiple reaction monitoring (MRM) methods, previously used for CK profiling on triple quadrupole mass spectrometers. This method offers improved mass accuracy and the complete product ion mass spectra (MS/MS) for compound de-termination with increased specificity and the sensitivity comparable with triple quadrupole instruments. The presented PRM approach was successfully applied to quantify 32 CKs in several biological samples.

Cytokinins (CKs) encompass a group of phytohormones, known to orchestrate many critical processes in plant development. Excluding Archaea, CKs are pervasive among all kingdoms, but much less is reported about their metabolism beyond plants. Recent evidence from mammalian tissues indicates the presence of six additional CK forms beyond the previously identified, single mammalian CK, N ⁶ ‐isopentenyladenosine (i ⁶ A). There is limited understanding of CK biosynthesis pathways in mammalian systems; therefore, human cervical cancer (HeLa) cells were used to further characterize CK processing by tracking the interconversion of CKs into their various structural derivatives in mammalian cells in a time‐course study. Through high‐performance liquid chromatography‐positive electrospray ionization‐tandem mass spectrometry (HPLC‐(+ESI)‐MS/MS), we document changes in the functional profiles of endogenous CKs in a human cell line following metabolism by HeLa cell cultures. The nucleotide CK fraction (iPRP) was found exclusively within the cell pellet (0.34 pmol/10 ⁶ cells), and the active free base (FB) form (iP) and riboside fraction (iPR) were found in greater abundance extracellularly (1.67 and 0.10 nmol/L respectively). For further confirmation, we demonstrate that HeLa cells metabolize an exogenously supplied CK, N ⁶ ‐benzyladenosine (BAR). In the HeLa culture supernatant, a 12‐fold decrease in BAR concentration was observed within the first 24 hours of incubation accompanied by a fivefold increase in the FB form, N ⁶ ‐benzyladenine (BA). These findings support the hypothesis that HeLa cells have the enzymatic pathways required for the metabolism of both endogenous and exogenous CKs.