Comparative Cell Biology Research Articles

Evolutionary origins of the lysosome-related organelle sorting machinery reveal ancient homology in post-endosome trafficking pathways

The major organelles of the endomembrane system were in place by the time of the last eukaryotic common ancestor (LECA) (~1.5 billion years ago). Their acquisitions were defining milestones during eukaryogenesis. Comparative cell biology and evolutionary analyses show multiple instances of homology in the protein machinery controlling distinct interorganelle trafficking routes. Resolving these homologous relationships allows us to explore processes underlying the emergence of additional, distinct cellular compartments, infer ancestral states predating LECA, and explore the process of eukaryogenesis itself. Here, we undertake a molecular evolutionary analysis (including providing a transcriptome of the jakobid flagellate Reclinomonas americana), exploring the origins of the machinery responsible for the biogenesis of lysosome-related organelles (LROs), the Biogenesis of LRO Complexes (BLOCs 1,2, and 3). This pathway has been studied only in animals and is not considered a feature of the basic eukaryotic cell plan. We show that this machinery is present across the eukaryotic tree of life and was likely in place prior to LECA, making it an underappreciated facet of eukaryotic cellular organisation. Moreover, we resolve multiple points of ancient homology between all three BLOCs and other post-endosomal retrograde trafficking machinery (BORC, CCZ1 and MON1 proteins, and an unexpected relationship with the "homotypic fusion and vacuole protein sorting" (HOPS) and "Class C core vacuole/endosomal tethering" (CORVET) complexes), offering a mechanistic and evolutionary unification of these trafficking pathways. Overall, this study provides a comprehensive account of the rise of the LROs biogenesis machinery from before the LECA to current eukaryotic diversity, integrating it into the larger mechanistic framework describing endomembrane evolution.

Preparation of Xenopus borealis and Xenopus tropicalis Egg Extracts for Comparative Cell Biology and Evolutionary Studies.

Cytoplasmic extracts prepared from eggs of the African clawed frog Xenopus laevis are extensively used to study various cellular events including the cell cycle, cytoskeleton dynamics, and cytoplasm organization, as well as the biology of membranous organelles and phase-separated non-membrane-bound structures. Recent development of extracts from eggs of other Xenopus allows interspecies comparisons that provide new insights into morphological and biological size variations and underlying mechanisms across evolution. Here, we describe methods to prepare cytoplasmic extracts from eggs of the allotetraploid Marsabit clawed frog, Xenopus borealis, and the diploid Western clawed frog, Xenopus tropicalis. We detail mixing and "hybrid" experiments that take advantage of the physiological but highly accessible nature of extracts to reveal the evolutionary relationships across species. These new developments create a robust and versatile toolbox to elucidate molecular, cell biological, and evolutionary questions in essential cellular processes.

Kinetics of Gene Expression Changes in Equine Fetal Interzone and Anlagen Cells Over 14 Days of Induced Chondrogenesis.

Within developing synovial joints, interzone and anlagen cells progress through divergent chondrogenic pathways to generate stable articular cartilage and transient hypertrophic anlagen cartilage, respectively. Understanding the comparative cell biology between interzone and anlagen cells may provide novel insights into emergent cell-based therapies to support articular cartilage regeneration. The aim of this study was to assess the kinetics of gene expression profiles in these skeletal cell lines after inducing chondrogenesis in culture. Interzone and anlagen cells from seven equine fetuses were isolated and grown in a TGF-β1 chondrogenic inductive medium. Total RNA was isolated at ten time points (0, 1.5, 3, 6, 12, 24, 48, 96, 168, and 336 h), and gene expression for 93 targeted gene loci was measured in a microfluidic RT-qPCR system. Differential transcriptional responses were observed as early as 1.5 h after the initiation of chondrogenesis. Genes with functional annotations that include transcription regulation responded to the chondrogenic stimulation earlier (1.5–96 h) than genes involved in signal transduction (1.5–336 h) and the extracellular matrix biology (3–336 h). Between interzone and anlagen cell cultures, expression levels of 73 out of the 93 targeted genes were not initially different at 0 h, but 47 out of the 73 genes became differentially expressed under the chondrogenic stimulation. While interzone and anlagen cells are both chondrogenic, they display clear differences in response to the same TGF-β1 chondrogenic stimulation. This study provides new molecular insight into a timed sequence of the divergent developmental fates of interzone and anlagen cells in culture over 14 days.

Tracing cell-type evolution by cross-species comparison of cell atlases

Cell types are the basic building units of multicellular life, with extensive diversities. The evolution of cell types is a crucial layer of comparative cell biology but is thus far not comprehensively studied. We define a compendium of cell atlases using single-cell RNA-seq (scRNA-seq) data from seven animal species and construct a cross-species cell-type evolutionary hierarchy. We present a roadmap for the origin and diversity of major cell categories and find that muscle and neuron cells are conserved cell types. Furthermore, we identify a cross-species transcription factor (TF) repertoire that specifies major cell categories. Overall, our study reveals conservation and divergence of cell types during animal evolution, which will further expand the landscape of comparative genomics.

Knockoff: Druggable Cleavage of Membrane Proteins.

Comparative cell biology relies on methods that disrupt protein function. Traditional approaches target the gene that encodes the protein of interest via conventional knockout (KO) methodology, conditional Cre-lox system, or recently, flexible protocols based on CRISPR/Cas9. However, these technologies lack precise temporal control (hours), whereby the slow half-lives of proteins may confound measurements, possibly resulting in misleading phenotypes. Targeting the protein itself bypasses issues pertaining to protein half-life, resulting in more acute disruption. An ideal system would enable controllable protein disruption, dependent on the presence or absence of a small molecule, with high temporal control achieved through washout/addition of the small molecule. Here, we outline the use of knockoff, a general method to disrupt membrane proteins based on the NS3/4A protease of the hepatitis C virus. This technique has been used in post-mitotic cells to study the function of long-lived integral membrane proteins and is suitable for the study of other membrane-bound proteins. Graphic abstract: Removal of the protease inhibitor induces cleavage from the membrane. General model of knockoff method. Inh, Inhibitor; POI, Protein of Interest; NS3/4A, Hepatitis C viral protease.

Symbiotic Regulatory Genes Controlling Nodule Development in Pisum sativum L.

Analyses of natural variation and the use of mutagenesis and molecular-biological approaches have revealed 50 symbiotic regulatory genes in pea (Pisum sativum L.). Studies of genomic synteny using model legumes, such as Medicago truncatula Gaertn. and Lotus japonicus (Regel) K. Larsen, have identified the sequences of 15 symbiotic regulatory genes in pea. These genes encode receptor kinases, an ion channel, a calcium/calmodulin-dependent protein kinase, transcription factors, a metal transporter, and an enzyme. This review summarizes and describes mutant alleles, their phenotypic manifestations, and the functions of all identified symbiotic regulatory genes in pea. Some examples of gene interactions are also given. In the review, all mutant alleles in genes with identified sequences are designated and still-unidentified symbiotic regulatory genes of great interest are considered. The identification of these genes will help elucidate additional components involved in infection thread growth, nodule primordium development, bacteroid differentiation and maintenance, and the autoregulation of nodulation. The significance of symbiotic mutants of pea as extremely fruitful genetic models for studying nodule development and for comparative cell biology studies of legume nodules is clearly demonstrated. Finally, it is noted that many more sequences of symbiotic regulatory genes remain to be identified. Transcriptomics approaches and genome-wide sequencing could help address this challenge.

Acute disruption of the synaptic vesicle membrane protein synaptotagmin 1 using knockoff in mouse hippocampal neurons.

The success of comparative cell biology for determining protein function relies on quality disruption techniques. Long-lived proteins, in postmitotic cells, are particularly difficult to eliminate. Moreover, cellular processes are notoriously adaptive; for example, neuronal synapses exhibit a high degree of plasticity. Ideally, protein disruption techniques should be both rapid and complete. Here, we describe knockoff, a generalizable method for the druggable control of membrane protein stability. We developed knockoff for neuronal use but show it also works in other cell types. Applying knockoff to synaptotagmin 1 (SYT1) results in acute disruption of this protein, resulting in loss of synchronous neurotransmitter release with a concomitant increase in the spontaneous release rate, measured optically. Thus, SYT1 is not only the proximal Ca2+ sensor for fast neurotransmitter release but also serves to clamp spontaneous release. Additionally, knockoff can be applied to protein domains as we show for another synaptic vesicle protein, synaptophysin 1.

Differential cell persistence is observed in the Arabidopsis female gametophyte during heat stress.

The central cell withstands heat stress better than the egg and antipodal cells. Insilco analysis of transcriptomic data identified several heat responsive genes which are central cell specific. Crop damage due to heat stress (HS) is a major cause of yield lost. Plants are particularly susceptible to negative effects of HS during gametophyte development and fertilization. Extensive studies have been performed on the male gametophyte under HS, but how the female gametophyte copes with HS is largely unknown. To learn how the different cell types of the female gametophyte reacts to HS, we studied unfertilized CDC123::H2B:YFP ovules. We found that the YFP-specific florescent signal persisted in the central cell during HS significantly more than the egg cell. We also found that the fluorescent signal persistence was the lowest in the antipodal cells. This finding suggests that the reaction of the female gametophyte to HS is rather unique and differentially mediated according to the cell's identity. In addition, mining through published transcriptomic datasets we found that several important heat stress responsive genes which are extremely upregulated during HS (more than 64-fold) are specifically expressed in the CC but not in the EC. Further research such as comparative transcriptomics and cell biology will likely shed more light on the phenomena reported here and increase our basic understandings about the ways sexual reproduction processes are affected by heat stress.

Genetic transformation of Spizellomyces punctatus, a resource for studying chytrid biology and evolutionary cell biology.

Chytrids are early-diverging fungi that share features with animals that have been lost in most other fungi. They hold promise as a system to study fungal and animal evolution, but we lack genetic tools for hypothesis testing. Here, we generated transgenic lines of the chytrid Spizellomyces punctatus, and used fluorescence microscopy to explore chytrid cell biology and development during its life cycle. We show that the chytrid undergoes multiple rounds of synchronous nuclear division, followed by cellularization, to create and release many daughter 'zoospores'. The zoospores, akin to animal cells, crawl using actin-mediated cell migration. After forming a cell wall, polymerized actin reorganizes into fungal-like cortical patches and cables that extend into hyphal-like structures. Actin perinuclear shells form each cell cycle and polygonal territories emerge during cellularization. This work makes Spizellomyces a genetically tractable model for comparative cell biology and understanding the evolution of fungi and early eukaryotes.

Comparative molecular cell biology of phototrophic euglenids and parasitic trypanosomatids sheds light on the ancestor of Euglenozoa.

Parasitic trypanosomatids and phototrophic euglenids are among the most extensively studied euglenozoans. The phototrophic euglenid lineage arose relatively recently through secondary endosymbiosis between a phagotrophic euglenid and a prasinophyte green alga that evolved into the euglenid secondary chloroplast. The parasitic trypanosomatids (i.e. Trypanosoma spp. and Leishmania spp.) and the freshwater phototrophic euglenids (i.e. Euglena gracilis) are the most evolutionary distant lineages in the Euglenozoa phylogenetic tree. The molecular and cell biological traits they share can thus be considered as ancestral traits originating in the common euglenozoan ancestor. These euglenozoan ancestral traits include common mitochondrial presequence motifs, respiratory chain complexes containing various unique subunits, a unique ATP synthase structure, the absence of mitochondria-encoded transfer RNAs (tRNAs), a nucleus with a centrally positioned nucleolus, closed mitosis without dissolution of the nuclear membrane and nucleoli, a nuclear genome containing the unusual 'J' base (β-D-glucosyl-hydroxymethyluracil), processing of nucleus-encoded precursor messenger RNAs (pre-mRNAs) via spliced-leader RNA (SL-RNA) trans-splicing, post-transcriptional gene silencing by the RNA interference (RNAi) pathway and the absence of transcriptional regulation of nuclear gene expression. Mitochondrial uridine insertion/deletion RNA editing directed by guide RNAs (gRNAs) evolved in the ancestor of the kinetoplastid lineage. The evolutionary origin of other molecular features known to be present only in either kinetoplastids (i.e. polycistronic transcripts, compaction of nuclear genomes) or euglenids (i.e. monocistronic transcripts, huge genomes, many nuclear cis-spliced introns, polyproteins) is unclear.

Joseph Thomas Bagnara (1929-2016), a pioneer of Pigment Cell Biology par excellence.

The international pigment cell research community lost one of its pioneers and pillars with the passing away of Professor Joseph T. Bagnara on October 2, 2016. Joe, as we, his students who became his friends, called him, obtained his Ph.D. in 1956 from the State University of Iowa (now known as University of Iowa). He spent his entire career at the University of Arizona, rising through the ranks to Professor and Chairman of the Department of General Biology. In 1992, he became Professor Emeritus. He rightly merits to be called the father of comparative pigment cell biology, as he focused most of his research on investigating the regulation of color change in different amphibians. He was a hard-core embryologist, zoologist, and endocrinologist. Joe taught the Embryology courses at the University of Arizona, and his Embryology text book was used by many professors in different universities. He was a scholar with a vision who desired to connect with other scientists internationally. He was twice a Fulbright Research Scholar, which provided him with the opportunity to do research in the Laboratoire d'Embryologie, Faculté des Sciences, Université de Paris, Paris, France, and later at the Stazione Zoologica di Napoli in Italy. He pioneered the research on the role of the pineal gland in regulating skin lightening and the identification of the different types of pigment cells in amphibians. He had a multidisciplinary approach to his research, using in vivo experiments, organ and tissue culture, electron microscopy as well as biochemistry to demonstrate the effects of different hormones and the role of different chromatophores in regulating rapid color changes in frogs. Many of his publications are classics in the pigment cell research literature. He collaborated with his colleagues at the University of Arizona, and also with many pigment cell researchers in Europe and Japan. Joe cherished his role as a teacher and mentor. He was always proud of his mentees and their achievements. On his retirement party, which was held in 1995 during the annual meeting of the PanAmerican Society for Pigment Cell Research in Kansas City, Kansas, his former students gathered to celebrate his scientific achievements. The group included Dr. Sally K. Frost-Mason, who was President of the PanAmerican Society for Pigment Cell Research and until recently the President of the University of Iowa, and John Taylor, who was Professor at Wayne State University in Detroit, Michigan. Joe should be acknowledged for founding, in 1987, the journal Pigment Cell Research (now Pigment Cell and Melanoma Research) and for serving as its first editor-in-chief. He was very fond and proud of this personal project, and we are grateful that he gave us this medium to communicate our science and expand the breadth of our specialty. Joe also played a major role in establishing the International Federation for Pigment Cell Societies and is one of the founders of the PanAmerican Society for Pigment Cell Research, which was conceived during the International Pigment Cell meeting in Tucson, Arizona, in 1986. Joe won many prestigious international and national awards, including the Myron Gordon Award from the IFPCS, and the Career Achievement Award from PASPCR. Joe wrote his autobiography “Unfinished Business: a Biologist in the Latter Half of the 20th Century,” which is a “must read” for all pigment cell researchers, as he narrates the history not only of his own career and travels but also of the evolution of pigment cell research worldwide and the establishment of the International Federation of Pigment Cell Societies. Those of us who had the honor and privilege to know Joe personally remember him not only as a kind mentor but mostly as a friend who remained in touch with us and always provided words of encouragement and support. On a personal note, thanks to Joe, I joined the Department of Dermatology at the University of Cincinnati in 1985 after graduating with a Ph.D. from the University of Arizona. He was the one who introduced me to James Nordlund, M.D., the Chairman of the department and my postdoctoral mentor at the time. Joe enjoyed life, loved the wilderness, was an avid athlete, and cared for his students and friends without limits. He was proud to know several languages and to have international friends and colleagues. He inspired us to work hard yet enjoy life to the fullest. “If I have seen further it is by standing on the shoulders of giants” (Isaac Newton). Mentors are persons who help shape our lives without molding us necessarily in their image. Joe, you are gone, but your legacy lives in all of us who knew you. Fond memories always remain, and we strive to pass the torch to our own students and mentees.

Comparative Cell Biology and Evolution of Annexins in Diplomonads.

Annexins are multifunctional, calcium-binding proteins found in organisms across all kingdoms. Most studies of annexins from single-celled eukaryotes have focused on the alpha-giardins, proteins assigned to the group E annexins, expressed by the diplomonad Giardia intestinalis. We have characterized the annexin gene family in another diplomonad parasite, Spironucleus salmonicida, by phylogenetic and experimental approaches. We constructed a comprehensive phylogeny of the diplomonad group E annexins and found that they are abundant across the group with frequent gene duplications and losses. The annexins of S.salmonicida were found to be related to alpha-giardins but with better-preserved type II Ca(2+) coordination sites. Two annexins were confirmed to bind phospholipids in a Ca(2+)-dependent fashion but with different specificities. Superresolution and confocal microscopy of epitope-tagged S.salmonicida annexins revealed localization to distinct parts of the cytoskeleton and membrane. The ultrastructural details of the localization of several annexins were determined by proximity labeling and transmission electron microscopy. Two annexins localize to a novel cytoskeletal structure in the anterior of the cell. Our results show that the annexin gene family is expanded in diplomonads and that these group E annexins are associated mostly with cytoskeletal and membrane structures. IMPORTANCE Annexins are proteins that associate with phospholipids in a Ca(2+)-dependent fashion. These proteins have been intensely studied in animals and plants because of their importance in diverse cellular processes, yet very little is known about annexins in single-celled eukaryotes, which represent the largest diversity of organisms. The human intestinal parasite Giardia intestinalis is known to have more annexins than humans, and they contribute to its pathogenic potential. In this study, we investigated the annexin complement in the salmon pathogen Spironucleus salmonicida, a relative of G.intestinalis. We found that S.salmonicida has a large repertoire of annexins and that the gene family has expanded separately across diplomonads, with members showing sequence diversity similar to that seen across kingdom-level groups such as plants and animals. S.salmonicida annexins are prominent components of the cytoskeleton and membrane. Two annexins are associated with a previously unrecognized structure in the anterior of the cell.

Evolutionary mechanisms for establishing eukaryotic cellular complexity

Through a comparative approach, evolutionary cell biology makes use of genomics, bioinformatics, and cell biology of non-model eukaryotes to provide new avenues for understanding basic cellular processes. This approach has led to proposed mechanisms underpinning the evolution of eukaryotic cellular organization including endosymbiotic and autogenous processes and neutral and adaptive processes. Together these mechanisms have contributed to the genesis and complexity of organelles, molecular machines, and genome architecture. We review these mechanisms and suggest that a greater appreciation of the diversity in eukaryotic form has led to a more complete understanding of the evolutionary connections between organelles and the unexpected routes by which this diversity has been reached.

The cell biology of the endocytic system from an evolutionary perspective.

Evolutionary cell biology can afford an interdisciplinary comparative view that gives insights into both the functioning of modern cells and the origins of cellular systems, including the endocytic organelles. Here, we explore several recent evolutionary cell biology studies, highlighting investigations into the origin and diversity of endocytic systems in eukaryotes. Beginning with a brief overview of the eukaryote tree of life, we show how understanding the endocytic machinery in a select, but diverse, array of organisms provides insights into endocytic system origins and predicts the likely configuration in the last eukaryotic common ancestor (LECA). Next, we consider three examples in which a comparative approach yielded insight into the function of modern cellular systems. First, using ESCRT-0 as an example, we show how comparative cell biology can discover both lineage-specific novelties (ESCRT-0) as well as previously ignored ancient proteins (Tom1), likely of both evolutionary and functional importance. Second, we highlight the power of comparative cell biology for discovery of previously ignored but potentially ancient complexes (AP5). Finally, using examples from ciliates and trypanosomes, we show that not all organisms possess canonical endocytic pathways, but instead likely evolved lineage-specific mechanisms. Drawing from these case studies, we conclude that a comparative approach is a powerful strategy for advancing knowledge about the general mechanisms and functions of endocytic systems.

Platelet Granule Exocytosis: A Comparison with Chromaffin Cells

The rapid secretion of bioactive amines from chromaffin cells constitutes an important component of the fight or flight response of mammals to stress. Platelets respond to stresses within the vasculature by rapidly secreting cargo at sites of injury, inflammation, or infection. Although chromaffin cells derive from the neural crest and platelets from bone marrow megakaryocytes, both have evolved a heterogeneous assemblage of granule types and a mechanism for efficient release. This article will provide an overview of granule formation and exocytosis in platelets with an emphasis on areas in which the study of chromaffin cells has influenced that of platelets and on similarities between the two secretory systems. Commonalities include the use of transporters to concentrate bioactive amines and other cargos into granules, the role of cytoskeletal remodeling in granule exocytosis, and the use of granules to provide membrane for cytoplasmic projections. The SNAREs and SNARE accessory proteins used by each cell type will also be considered. Finally, we will discuss the newly appreciated role of dynamin family proteins in regulated fusion pore formation. This evaluation of the comparative cell biology of regulated exocytosis in platelets and chromaffin cells demonstrates a convergence of mechanisms between two disparate cell types both tasked with responding rapidly to physiological stimuli.

Stem cells in the context of evolution and development

Stem cells in the context of evolution and development

The Naegleria genome: a free-living microbial eukaryote lends unique insights into core eukaryotic cell biology

Naegleria gruberi, a free-living protist, has long been treasured as a model for basal body and flagellar assembly due to its ability to differentiate from crawling amoebae into swimming flagellates. The full genome sequence of Naegleria gruberi has recently been used to estimate gene families ancestral to all eukaryotes and to identify novel aspects of Naegleria biology, including likely facultative anaerobic metabolism, extensive signaling cascades, and evidence for sexuality. Distinctive features of the Naegleria genome and nuclear biology provide unique perspectives for comparative cell biology, including cell division, RNA processing and nucleolar assembly. We highlight here exciting new and novel aspects of Naegleria biology identified through genomic analysis.

Small Cells—Big Future

Small Cells—Big Future

Functional angiotensin‐converting enzyme 2 is expressed in human cardiac myofibroblasts

The renin-angiotensin system (RAS), in particular angiotensin II, plays an important role in cardiac remodelling. Angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) are key players in the RAS and act antagonistically to regulate the levels of angiotensin II. In this study, we reveal the functional expression of ACE2 in human cardiac myofibroblasts, cells that are essential to the maintenance of normal cardiac architecture and also play a key role in myocardial remodelling. The observed reciprocal expression of ACE and ACE2 in these cells may reflect the possible opposing activity of these two enzymes. In this study, we demonstrate the presence of ACE2 as an ectoenzyme and reveal that ACE2 undergoes phorbol-12-myristate-13-acetate-inducible ectodomain shedding from the membrane. When cells were exposed to a number of pathophysiological stimuli, modulation of ACE2 levels was not detected. Importantly, whilst we found ACE2 to be expressed constitutively in cardiac myofibroblasts there were no detectable levels in either vascular smooth muscle cells or vascular endothelium, indicating that ACE2 expression is not ubiquitous. In paraffin sections of atrial appendage tissue, we observed a distinct staining pattern for ACE2 which appeared different from that of ACE. In conclusion, this study is the first to report co-expression of ACE and ACE2 in human cardiac myofibroblasts and may therefore present a model primary system for study of the comparative cell biology of ACE2 and ACE and their potentially opposing roles in myocardial remodelling.

RNA expression in a cartilaginous fish cell line reveals ancient 3' noncoding regions highly conserved in vertebrates.

We have established a cartilaginous fish cell line [Squalus acanthias embryo cell line (SAE)], a mesenchymal stem cell line derived from the embryo of an elasmobranch, the spiny dogfish shark S. acanthias. Elasmobranchs (sharks and rays) first appeared >400 million years ago, and existing species provide useful models for comparative vertebrate cell biology, physiology, and genomics. Comparative vertebrate genomics among evolutionarily distant organisms can provide sequence conservation information that facilitates identification of critical coding and noncoding regions. Although these genomic analyses are informative, experimental verification of functions of genomic sequences depends heavily on cell culture approaches. Using ESTs defining mRNAs derived from the SAE cell line, we identified lengthy and highly conserved gene-specific nucleotide sequences in the noncoding 3' UTRs of eight genes involved in the regulation of cell growth and proliferation. Conserved noncoding 3' mRNA regions detected by using the shark nucleotide sequences as a starting point were found in a range of other vertebrate orders, including bony fish, birds, amphibians, and mammals. Nucleotide identity of shark and human in these regions was remarkably well conserved. Our results indicate that highly conserved gene sequences dating from the appearance of jawed vertebrates and representing potential cis-regulatory elements can be identified through the use of cartilaginous fish as a baseline. Because the expression of genes in the SAE cell line was prerequisite for their identification, this cartilaginous fish culture system also provides a physiologically valid tool to test functional hypotheses on the role of these ancient conserved sequences in comparative cell biology.