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Evolutionary cell biology makes use of genomics, bioinformatics, and cell biology of non-model eukaryotes to provide new avenues for understanding basic cellular processes. These mechanisms have contributed to the genesis and complexity of organelles, molecular machines, and genome architecture. Understanding these mechanisms will help us to port knowledge derived in model systems to less studied organisms of agricultural, environmental, or medical relevance.
Evolutionary cell biology makes use of genomics, bioinformatics, and cell biology of non-model eukaryotes to provide new avenues for understanding basic cellular processes. These mechanisms have contributed to the genesis and complexity of organelles, molecular machines, and genome architecture. Understanding these mechanisms will help us to port knowledge derived in model systems to less studied organisms of agricultural, environmental, or medical relevance.
Evolutionary cell biology makes use of genomics, bioinformatics, and cell biology of non-model eukaryotes to provide new avenues for understanding basic cellular processes. These mechanisms have contributed to the genesis and complexity of organelles, molecular machines, and genome architecture. Understanding these mechanisms will help us to port knowledge derived in model systems to less studied organisms of agricultural, environmental, or medical relevance.
complexity Fred D. Mast 1,2,3 , Lael D. Barlow 1 , Richard A. Rachubinski 1 , and Joel B. Dacks 1 1 Department of Cell Biology, University of Alberta, Edmonton, Alberta T6G 2H7, Canada 2 Seattle Biomedical Research Institute, 307 Westlake Avenue North, Seattle, WA 98109-5240, USA 3 Institute for Systems Biology, 401 Terry Avenue North, Seattle, WA 98109-5219, USA Through a comparative approach, evolutionary cell biol- ogy makes use of genomics, bioinformatics, and cell biology of non-model eukaryotes to provide new ave- nues for understanding basic cellular processes. This approach has led to proposed mechanisms underpin- ning 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 com- plexity 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. Bringing together cell biology and evolutionary biology The emergence of the eukaryotic state nearly 2 billion years ago transformed life on Earth. Efforts to unravel the evolutionary mechanisms that have shaped, and con- tinue to shape, eukaryotic cells are beginning to address this monumental evolutionary shift. Understanding these mechanisms will help us to make conceptual connections between the cell biology of taxonomically diverse modern eukaryotes, porting knowledge derived in model systems to less studied organisms of agricultural (e.g., crops, plant pathogens), environmental (e.g., aquatic primary produ- cers like haptophytes and diatoms), or medical (e.g., para- sites such as Plasmodium falciparum, the causative agent of malaria) relevance. This broad comparative approach known as evolutionary cell biology (see Glossary) facili- tates the generation of hypotheses that attempt to explain the cell biological functions shared among the full range of eukaryotes. This approach has been applied successfully to many aspects of the eukaryotic cell (e.g., [1]). The combination of ultrastructure and molecular cell biology with genomic data from a sampling of organisms spanning the taxonomic breadth of eukaryotes [2,3] (Figure 1) has provided a wealth of knowledge regarding the evolution of eukaryotic cell biology and its diversity. From the perspective of a cell biologist, this wealth of data allows the integration of established evolutionary theory with the study of cellular mechanisms. Review Glossary Complexity: a measure of the number of components and interactions of one system relative to another equivalent system. Endosymbiosis (primary): the process whereby a prokaryotic cell (endosym- biont) is incorporated into the cytoplasm of a eukaryotic cell (host), with a relationship being established via metabolic integration and EGT such that neither partner can survive on its own. Endosymbiosis (secondary): the same process as primary endosymbiosis except that the endosymbiont is a eukaryotic cell possessing a primary plastid. The process can be extended to tertiary endosymbiosis (the endosymbiont is a cell possessing a secondary plastid) and serial secondary endosymbiosis (a lineage possessing one type of secondary plastid replaces its secondary plasmid with a secondary plastid of a different lineage). Endosymbiotic gene transfer (EGT): a special case of horizontal gene transfer (see below), whereby the gene in question is acquired by the host lineage from the genome of the endosymbiont. Evolutionary cell biology: an emerging discipline that incorporates compara- tive perspectives and techniques from cell biology, protistology, molecular evolution, and mathematical evolutionary theory to address questions of the origins and diversity of cells. First eukaryotic common ancestor (FECA): the cell (or population of cells) belonging to the lineage that gave rise to the modern line of eukaryotes at the earliest point at which it possessed cell biological features distinct from those in prokaryote-like cells. Although this organism is deduced to have existed, a useful way to treat the FECA is as a theoretical reconstruction with the traits defining it as an exciting open research question. Horizontal gene transfer: the acquisition of a gene by a genome froma source other than the immediate parental lineage. Last eukaryotic common ancestor (LECA): the cell (or population of cells) belonging to the lineage that gave rise to the modern line of eukaryotes at the latest point at which the various descendent lineages diverged to leave the extant eukaryotic lineages. Again, this concept is most useful as a theoretical reconstruction or reference point to assess the antiquity of various cell biological features. Monophyletic: a group is considered monophyletic when it encompasses all descendants of a single ancestor. Paraphyletic: a group is considered paraphyletic when it encompasses some, but not all, descendants of a single ancestor. Paralog: genes that are the result of a gene duplication process. Selection: the process by which a factor (including the presence of another organism) presents a circumstance that results in the preferential death of some organisms in the environment over others. 0962-8924/$ see front matter 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tcb.2014.02.003 Corresponding author: Dacks, J.B. (dacks@ualberta.ca). Keywords: constructive neutral evolution; endosymbiosis; evolutionary cell biology; organelle paralogy hypothesis; protocoatomer; transfer-window hypothesis. Trends in Cell Biology, July 2014, Vol. 24, No. 7 435 Signicant progress has been made in revealing the mechanisms underpinning the emergence of complexity in the eukaryotic cell. The number and sophistication of internal compartments in eukaryotes distinguish them from prokaryotes and it is this sophistication that trans- lates into vastly greater complexity in eukaryotic cellular conguration, which is measured by the number of com- ponents in a system and the number of interactions be- tween them [4,5] compared with another, equivalent system (e.g., cell versus cell). There is general agreement from both molecular phylogenetic analyses [6,7] and evi- dence from the fossil record ([8,9], but see [10]) that pro- karyotes predated eukaryotes. This evidence suggests that eukaryotes must have arisen from a state resembling that of a prokaryote; that is, that the acquisition of organelles and complex cellular machines in eukaryotes must be explained from a cellular state lacking the extensive pres- ence of these features. Evolution from less complexity (prokaryote state) to increased complexity of cellular ar- chitecture (eukaryote state) can be divided into at least three successive evolutionary stages (Figure 1). First, a transition from an organism lacking some or all internal membranes to an organism that possesses some cellular features that dene it as eukaryotic [i.e., a rst eukaryotic common ancestor (FECA)] must occur. Next, the organism must transition from the FECA to a last eukaryotic com- mon ancestor (LECA). Finally, evolution and divergence after the LECA must occur to form the major lineages of extant eukaryotes [2,11]. Research on the prokaryote-to-eukaryote transition (e.g., [1]) has reconstructed a surprisingly sophisticated LECA possessing a well established actin/tubulin cytoskel- eton, an elaborate endomembrane system, a nucleus, mitochondria, and machinery for processes such as intron splicing and meiosis (Figure 1). Furthermore, at least one additional major cellular innovation has inuenced post- LECA increases in complexity: the acquisition of plastids. Therefore, an important question for evolutionary cell biology is what mechanisms drive increased cellular com- plexity at the level of molecular machines and the forma- tion of organelles. Here we review the progress that has been made in addressing this question. Mechanisms for organelle acquisition Before 1974, the null hypothesis for the evolution of inter- nal membrane-bound compartments (organelles) within the eukaryotic cell was an autogenous process; that is, eukaryotic cells are built in a stepwise manner from indi- vidual building blocks present in the pre-eukaryotic ances- tor [12,13]. However, evidence demonstrating that both mitochondria and chloroplasts are of bacterial origin (en- dosymbiosis) [14,15] shifted the theoretical basis of the eukaryotic evolutionary eld such that both endosymbiotic and autogenous explanations are now viable alternatives to entertain when addressing the origins of a eukaryotic organelle. Our scientic understanding of both mecha- nisms is maturing, with endosymbiosis being far better understood. Below, we discuss the current standing on the emergence of complexity of eukaryotic cells through endo- symbiosis and autogenous processes. Endosymbiosis Endosymbiosis is the incorporation and residence of one organism, the endosymbiont, inside another, the host. Beyond the familiar examples of endosymbiosis (i.e., the chloroplast of plants and the nearly ubiquitous mitochon- drion), a diverse array of organisms with organelles de- rived from additional endosymbiotic events also exists Prokaryote First eukaryoc common ancestor (FECA) Last eukaryoc common ancestor (LECA) Opisthokonta Amoebozoa Excavata Archaeplasda SAR CCTH Extant eukaryoc lineages (A) (B) TRENDS in Cell Biology Figure 1. Retracing the emergence of modern eukaryotes. (A) The emergence of eukaryotes can be divided into at least three successive evolutionary stages: (i) transition from a prokaryote-like starting point to the first eukaryotic common ancestor (FECA); (ii) transition from the FECA to the last eukaryotic common ancestor (LECA); and (iii) evolution and divergence after the LECA to form the major lineages of eukaryotes as we know them today [2,11]. (B) After the LECA, six extant eukaryotic lineages (Box 1) have been characterized [11]. These are the Opisthokonta, Amoebozoa, Excavata, and Archaeplastida, the stramenopiles, alveolates and rhizarians (SAR), and the contentious grouping of cryptomonads, centrohelids, telonemids, and haptophytes (CCTH). Cartoon images of representative organisms fromeach supergroup are not to scale. Review Trends in Cell Biology July 2014, Vol. 24, No. 7 436 (Figure 2A). Recent cell biological and genomic studies of these organisms have revealed much about the mechanism of endosymbiosis. Indeed, one reason why endosymbiosis is better understood than autogenous mechanisms of organ- elle acquisition is the wealth of endosymbiotic intermedi- ates available for study (e.g., [16]). Recent and independent occurrences of endosymbiosis have revealed the earliest stages of the process, including several examples of prima- ry (e.g., Paulinella) and secondary (e.g., Hatena) plastid- derived organelles as well as transiently acquired plastids termed kleptoplasts [16]. Other examples of where the host and symbiont are at the beginning of their integration include dinotoms, algae wherein the host lineage is a dinoagellate that possesses a minimally reduced diatom endosymbiont. Recent work has begun to uncover the extent and nature of their organellar and metabolic inte- gration [17]. Genome sequencing of organisms such as the cryptophyte and chlorarachniophyte algae, whose photo- synthetic organelle contains both a secondary plastid ge- nome and the remnant of the red or green algal nuclear genome (nucleomorph) and cytoplasm [18], have also allowed examination of genome reduction and cellular integration in endosymbiosis (Box 2). Because photosyn- thesis has been gained, stolen and co-opted throughout the history of eukaryotes, the acquisition of plastids has been a particularly useful model for understanding the early stages of endosymbiosis. However, a rare example of a potential secondary mitochondrial endosymbiont has re- cently been described by genomic methods [19]. The sh pathogen Neoparamoeba contains what appears to be an intracellular symbiont related to Ichthyobodo necator, a kinetoplastid. Although the atypical mitochondrion of this symbiont occupies nearly half of its cytoplasmic volume, the extent to which the endosymbiont has progressed to become an organelle is unclear. The nuclear genome of the symbiont does not appear to have undergone extensive reduction compared with that of other kinetoplastids. All of these examples help focus the question of how reduced an endosymbiont has to be for it to be considered an organelle and no longer an organism. At the other extreme of endosymbiotic integration exist organelles apparently reduced from the canonical eukary- otic state, such as the non-photosynthetic apicoplasts of apicomplexans [16] and the hydrogenosomes and mito- somes, some of which no longer possess organellar gen- omes. Initially these latter organelles were seen as distinct classes; however, recent studies have clearly established them as derivatives of mitochondria and found various intermediates possessing aerobic or anaerobic metabo- lisms and different genomic organizations (e.g., [20]). The range of genomic and cytoplasmic minimalization found for endosymbiotically derived organelles raises the question of what mechanism determines and limits the extent of this reductive trend in any given lineage. The passage of time cannot explain this reduction because a wide range of reduction is observed in organelles clearly derived from the same founding event (e.g., the mitochon- drion). However, it was proposed that because the main Box 1. Eukaryotic diversity Eukaryotic diversity (Figure 1B in main text) is currently divided into six large taxonomic groupings, or supergroups [2]. The Opistho- konta encompasses the lineages of animals and fungi, as well as their single-celled relatives. The Amoebozoa houses a diversity of amoeboid lineages with, and without, flagellated stages. It includes the pathogens Balamuthia, Acanthamoeba, and, most famously, Entamoeba histolytica, the causative agent of amoebic dysentery. The Opisthokonta and Amoebozoa are united in large-scale molecular phylogenetic analyses and thought to represent a monophyletic grouping, named the Amorphea [2]. The Archae- plastida incorporates the lineages of red algae, green algae (including land plants), and the glaucophytes, which are derived from a single founding primary-endosymbiotic event. The SAR clade unites the seemingly disparate lineages of stramenopiles (diatoms, brown algae, and the causative agent of the Irish Potato Famine, Phythophthora) and alveolates (ciliates like Paramecium, the dinoflagellates that cause red tides, and apicomplexans such as Plasmodium, which causes malaria). The supergroup Excavata includes important disease-causing agents such as Trypanosoma, Leishmania, Giardia, and Trichomonas, as well as their free-living, or nonpathogenic, relatives. Finally, the CCTH supergroup currently contains the lineages of cryptophytes, centrohelids, telonemids, and haptophytes; however, the most recent large-scale molecular- evolutionary analyses have cast doubt on the unity of these in a single group [3] and the CCTH should be treated as tentative at best. Box 2. Endosymbiosis The types of endosymbiosis are classified based on the nature of the host and of the endosymbiont. The simplest form, or primary endosymbiosis, involves a eukaryotic host and a bacterial endo- symbiont (Figure 2A in main text). Two such primary events have been transformative in the history of eukaryotes and involved the incorporation of an a-proteobacterium and a cyanobacterium to give rise to mitochondrion- and plastid-derived organelles, respec- tively. Both events are known to have occurred early in eukaryotic history, with the mitochondrial event now convincingly shown to have predated the LECA [20]. A primary plastid endosymbiosis is very likely to have occurred at the base of the Archaeplastida lineage, conferring photosynthetic capacity and giving rise to all red and green algae and land plants. The photosynthetic ability was clearly advantageous, as it spawned the subsequent evolution of complex plastids [16] through secondary and tertiary endosym- bioses (Figure 2A in main text). As a mechanism, the process of endosymbiosis can be divided into initiation and integration. Initiation may stem from various possible microbial associations, including mutualistic exchange of metabolites, intracellular invasion of the host by a parasite, or predatory ingestion of an eventual endosymbiont by a phagotroph. After initiation, the success of the resulting chimera depends on the ability to synchronize the cell growth and division cycles of the host and endosymbiont. In all cases, gradual transfer of genetic material from the endosymbiont genome to the host genome promotes this synchronization (Figure 2B in main text). This ratchet-like mechan- ism of EGT drives the establishment of an obligate relationship between the endosymbiont and its host. After acquiring an endosymbiont, the organism has two genomes, one in the nucleus and one in the endosymbiont (Figure 2B, i in main text). Whether by lysis or by improper fission and fusion events of the endosymbiont during replication, endosymbiont DNA released into the cytoplasm can be integrated into the host genome (Figure 2B, ii in main text). With an endosymbiont gene now encoded and expressed by the host, it must be successfully retargeted to the endosymbiont (Figure 2B, iii in main text). When this occurs, the endosymbiont copy is redundant and sustains mutational decay, and the endosymbiont genome is reduced (Figure 2B, iv in main text). The directionality imposed by this transfer results in an iterative ratchet- like mechanism. The window of opportunity permitting EGT remains open until only a single endosymbiont genome remains (Figure 2B, v and vi in main text) [16,20]. Loss of genes, coincident with loss of function, in the organism-to-organelle transition is also a major source of genome reduction [16,20]. Review Trends in Cell Biology July 2014, Vol. 24, No. 7 437 Independent acvity Binding and presuppression Mutaon and dependence Ratchet-like increase in dependence Acvity A Factor A Factor A Factor B Acvity A Factor A Factor B Acvity A Factor A Factor B Acvity A (i) (i) (ii) (iii) (ii) (iii) (iv) x y z x y z x y z x y z x y z x y z (v) x x y z x y z (vi) x y z y z y z y z x z x y Endosymbiosis (A) (B) (C) (D) EGT and transfer window hypothesis Organelle paralogy hypothesis Construcve neutral evoluon Primary Secondary Terary (i) (ii) (iii) (i) (ii) (iii) (iv) TRENDS in Cell Biology Figure 2. Mechanisms of cellular evolution. (A) The variety of plastids arising from iterative acquisition of photosynthetic endosymbionts. (i) Primary endosymbiosis is established following engulfment of a cyanobacterium (green) by a eukaryotic host cell. A similar primary endosymbiotic process would also have produced the mitochondrion from a proteobacterium. (ii) In secondary plastid endosymbiosis, a green or red algal cell is engulfed by a new host cell. (iii) This process is repeated in tertiary endosymbiosis. Although primary, secondary, and tertiary endosymbioses are conceptually interconnected, they are not consecutive steps of a single colonization. (B) The steps of endosymbiotic gene transfer (EGT) from a newly acquired endosymbiont: lysis of the endosymbiont and (ii) transfer of the gene to the host nucleus; (iii) retargeting and (iv) endosymbiont-encoded gene loss; and (v) repetition until (vi) a single endosymbiont remains. (C) The organelle-paralogy hypothesis (OPH). (i) Different protein families interact cooperatively to specify organelle-defining properties such as tethering, docking, fission, or fusion. (ii) Specificity-encoding protein families evolve by gene duplication and divergence, as represented by this hypothetical phylogeny. (iii) Increases in the complexity of specificity-encoding protein families are mirrored by increases in the complexity of the membrane-trafficking system. Paralogs of the specificity-encoding protein family reside in and have their effect on distinct compartments. Modified from[59]. (D) A generalized outline of constructive neutral evolution (CNE). (i) Protein factor A possesses a given activity. (ii) Through randomsteric collisions, a stochastic interaction with a separate factor B occurs that has little or no effect on the activity of factor A. (iii) A mutation (represented by a yellow star) occurs in factor A that reduces its activity, but due to the interaction of factor A with factor B, the mutation is suppressed and the activity of factor A is maintained at near-original levels. This could be due to stabilization of the structure of factor A, masking of its charge or exposed hydrophobic residues, or altered localization of factor A allowing better access to its substrate. (iv) Subsequent mutations of the original factor A, and compensatory mutations in the interacting factor B, further integrate factor B in the activity of factor A via a ratchet-like mechanism that may also lead to the recruitment of additional factors. Review Trends in Cell Biology July 2014, Vol. 24, No. 7 438 mechanism of DNA transfer to the host nucleus comes from lysed organelles, the rate of transfer is proportional to the copy number of the endosymbiont in the cell (Figure 2B). This idea became known as the transfer-window hypothe- sis [21], which implies that transfer cannot continue once the number of organelles has reached a single copy. Indeed, experimental [22] and comparative [23] genomic analyses revealed far fewer transfers from plastids to nuclear gen- omes in organisms possessing a low plastid copy number. In addition, the nuclear genomes of a cryptophyte and of a chlorarachniophyte alga, each possessing a single nucleo- morph, have been sequenced and were reported in 2012 [24]. These ndings revealed a complete lack of recent DNA transfer from either the plastid or the nucleomorph ge- nome despite evidence of transfer from mitochondria, which is consistent with a reduction to a single organelle that is responsible for halting endosymbiotic gene transfer (EGT) and hence organelle reduction. Endosymbiosis has repeatedly allowed for increased overall complexity in eukaryotic cells compared with their pre-merged state. Ironically, because the cell is at its most complex state immediately after endosymbiosis begins, with integration progressing principally via EGT or gene loss, the process of endosymbiosis actually involves decreases in complexity. Autogenous (non-endosymbiotic) organelles Although endosymbiosis has undoubtedly been a powerful force in building some aspects of eukaryotic cellular com- plexity, it does not explain them all. A simpler, alternative explanation for the origin of organelles delimited by a single lipid bilayer and devoid of genetic material is that they are autogenous. The organelles most commonly proposed to have an autogenous origin are those of the membrane-trafcking system, including the endoplasmic reticulum (ER), Golgi apparatus, endosomes, and plasma membrane [25]. Although these endomembrane organelles are dynamically connected to one another, they are main- tained as distinct compartments through the action of membrane trafcking machineries such as Rabs, SNAREs, coatomer, and adaptin (AP) complexes [26]. These speci- city-encoding protein families have different members that perform the same function (e.g., inducing membrane curvature or facilitating membrane fusion) at distinct locations within the membrane-trafcking system [26]. Although each protein family could play an individual role, part of the information encoding specicity in membrane trafcking appears to result from combinatorial protein protein interactions between members of the different families [27]. Comparative genomic and phylogenetic anal- yses of these various protein families have revealed details of their primary diversication by gene duplication (e.g., [1]). Surprisingly, the duplications giving rise to paralogs of the various specicity-encoding proteins associated with each cellular location occurred before the LECA. However, examination of the endocytic paralogs of the SNARE, Rab, and AP families revealed a pattern whereby some organ- elle-specic paralogs had not duplicated before the LECA, with parallel duplications occurring instead in lineages after the LECA [28]. These patterns provide an under- standing of the timing of these events and suggest a possible mechanism underpinning them, which is formal- ized in the organelle-paralogy hypothesis (OPH) [28,29]. The OPH (Figure 2C) proposes that a set of specicity- encoding proteins with complementary functions that de- ne organelle properties produce sets of interacting para- logs by undergoing duplications. Through coevolution, these sets of specicity-encoding proteins accumulate mutations that x their specic functional binding, thus dening separate organelles [30]. Iterations of this process could therefore account for the array of organelles in the endomembrane systems of extant eukaryotes that arose via differentiation from an original prototypical internal compartment in the FECA. Recently, the OPH has been tested by computer simu- lation. Mathematical modeling of specicity-encoding genes in populations of vesicles showed that gene duplica- tion and differential interactions between paralogs pro- duced novel vesicular compartments [31]. The OPH further predicts that the order of evolutionary emergence for each member of a specicity-encoding protein family should correspond to the order of emergence of the different organelles they dene and on which they have effect. Two recent studies have reported phylogenetic resolution for important specicity-encoding protein families, thereby allowing hypotheses to be proposed based on empirical evidence regarding an order of evolutionary emergence beyond the establishment of extensive complexity in mem- brane trafcking in the LECA. AP complexes aid in sorting the vesicular trafc between organelles found between and including the plasma membrane and the trans-Golgi net- work [32,33]. Comparative genomic and phylogenetic anal- ysis resolved the order of emergence of the members of the AP complex family, with AP3 and AP5 rst diverging from the remaining AP complexes, followed by AP4 and AP1/2 [32]. Based on their known locations of action, this order suggests that adaptins rst acted at an organellar inter- face between the secretory system and the phagocytic system, before the establishment of the trans-Golgi net- work. In addition, recent evidence provides clues to the conservation among the Rab family of GTPases, which are molecular switches involved in specifying organelle iden- tity in the membrane-trafcking system [34]. Although it is well established that Rab GTPases are ancient and that the LECA possessed a large complement of such proteins [35], the extent to which Rab families are conserved remained unknown. Rigorous homology searching resulted in the expansion of the Rab complement in LECA to 15 subfamilies [36]. However, robust phylogenetic resolution between the paralogs of the Rab gene families increased the estimated number of Rab subfamilies in the LECA to between 19 and 23 [37]. Surprisingly, this analysis also revealed two ancient sets of Rabs, one inferred to be involved in exocytosis and one predominantly in endocyto- sis, potentially reecting the earliest establishment of these pathways. As improved comparative and phyloge- netic methods are applied to other trafcking families, it will be important to compare the evolutionary patterns that emerge and to delve further into events pre-LECA. Although the OPH is a mechanism for evolving in- creased compartment number and specialization within an organellar system, it is currently limited to the Review Trends in Cell Biology July 2014, Vol. 24, No. 7 439 membrane-trafcking system. However, an idea that com- plements the OPHis the protocoatomer hypothesis, which proposes, based on protein-structural evidence, that ho- mology exists between the membrane deformation com- ponents of vesicular trafcking and the nuclear pore [38]. Specically, proteins integrated into the COP I, COP II, clathrin, and nuclear pore complexes share a structure of b-propellers followed by a-solenoid domains. These pro- teins are suggested to be homologous and therefore de- rived from a single ancestral protocoatomer protein [39]. Recent analyses have also rmly established relation- ships between protocoatomer-derived proteins of the intraagellar complex [40]. These proteins, which are dispersed throughout the cell and essential for organ- elle-specic functions, appear to have expanded along with their organelles via the process described in the OPH. Therefore, the overlap between the two hypotheses extends the mechanism of autogenous organelle evolution to potentially all organelles for whicha non-endosymbiotic origin appears likely. Examples exist of organelles whose origins blur the divisions of autogenous and endosymbiotic organellar evo- lution. The origin of the peroxisome has been contentiously explained by both mechanisms. Although the evidence, both functionally [41] and evolutionarily [42], strongly favors an autogenous origin for peroxisomes, there have undoubtedly been, and continue to be, molecular and functional interactions between peroxisomes and orga- nelles of endosymbiotic origin, notably the mitochondrion [43]. Many proteins that localize to the peroxisome are encoded by genes of bacterial origin and function in meta- bolic processes shared with mitochondria (e.g., fatty-acid oxidation). Determining how endosymbiotic organelles have become integrated within the cell and interact with non-endosymbiotically derived systems is an emerging area of investigation for cell biology and evolutionary cell biology. Work in the past few years has uncovered several protein complexes mediating protein, lipid, and ion trans- port between the ER and mitochondria [44] and it was recently shown that protein complexes bridging the ER and mitochondria in fungi are more widely present in eukaryotes than previously suspected [45,46]. Constructive neutral evolution (CNE) Evolutionary processes are not limited to the organellar level. Individual cellular machines in the eukaryote (e.g., ribosomes, proteasomes) also show increased complexity over their prokaryotic counterparts. In some cases, this increased complexity could result in new functions, pro- viding a selective advantage to the eukaryotic cell. How- ever, the role of selection as the only driver in the evolution of complexity is increasingly being questioned. The theory of CNE [47] posits that many biological phenomena can arise, or be elaborated on, by neutral evolutionary processes that promote increased complexity without additional functionality [48]. CNE is predicated on an idea of presuppression (Figure 2D); that is, interactions between factors that are the initial result of random colli- sions or cytosolic overcrowding and that minimally affect function [49] may become stabilized due to random muta- tion in a factors partner or in both factors. On their own, these mutations may be slightly deleterious for the origi- nal function, but if binding of the partner restores func- tionality, the interaction becomes xed. Therefore, the mutation is not selective in the traditional sense but needs to be sufciently compensatory to avoid negative selection and to allow the organism to survive. These mutations may be extremely rare; nevertheless, once established they result in a ratchet that promotes tighter binding and, potentially, recruits other factors. These interactions could involve protein interactions with nearly any mole- cule or surface in the cell (e.g., diffusible small molecules, cellular membranes). Among these biological phenomena, the origin of the spliceosome has been proposed to require CNE [47,48]. Comparative genomic studies of spliceosomal components have demonstrated that the spliceosome is a eukaryotic innovation that was present in its highly elaborate state before the LECA [50]. Comprising well over 100 different protein and RNA components, the spliceosome is a candi- date for one of the most complex cellular machines in existence. However, it has long been appreciated that the underlying essential process could have evolved from a simple self-splicing group II-class intron. Rather than being the result of selective forces, the spliceosome is best explained as a product of CNE whereby mutations in the self-splicing RNA molecule were suppressed through a pre- existing interaction with a RNA or RNA/protein complex [47,48]. As protein and RNA components accumulated over time, the basic function of splicing remained unaltered. A recently well elaborated example involving experi- mental testing of hypothesized CNE processes is the vacu- olar V 0 -ATPase ring of yeast [49,51]. Although the ancestor of the yeast V 0 -ATPase ring comprises two subunits, sev- eral yeasts require three subunits, with the third subunit resulting from an ancient gene duplication that was fol- lowed by gene suppression. To verify this sequence of events, investigators reconstructed the common ancestral gene of extant two-subunit rings and three-subunit rings and revealed specic suppressive interactions required to enforce the adoption of the three-subunit system [51]. When suppression succeeds, the system, because of this dependency, is more complex; however, the net effect of the increased complexity remains neutral in that no altera- tions in the cells ability to produce the phenotype have occurred. Therefore, CNE allows the accumulation of greater complexity combined with a dilution of responsi- bility for maintaining a phenotype among multiple factors. Ironically, this dilution, via redundant functionality of components, would reduce the risk of negative selection on a single mutational target and, as such, the CNE mechanism itself may be under positive selective pressure [48]. Concluding remarks The above overview was organized into processes acting at the level of the organelle or at the level of the underlying molecular complex, but such divisions are by no means absolute. Molecular machineries clearly cooperate to build and dene organelles. At the same time, the compartmen- talization of specic molecular machineries within a given organelle limits the range of proteins with which these Review Trends in Cell Biology July 2014, Vol. 24, No. 7 440 molecular machines will frequently interact and thereby increases the opportunities for distinct environments that would lead to complexity via CNE mechanisms. At present, tremendous opportunities exist for the ad- vancement of evolutionary cell biology as a discipline. While the eld brings evolutionary biology from the popu- lation and the large organism down to the scale of the cell, it also brings a comparative approach over species and space to cell biologists focused on specic organisms or organelles. However, there is also a potential for miscon- ceptions. In many ways, the study of cell biology shares conceptual commonalities with the discipline of reverse engineering [52]. Cell biology is typically understood from a reductionist approach whereby the cell is disassembled, both conceptually and physically, into its components (proteins, organelles, and complexes) and then laid out, manipulated, and understood. Therefore, it is unsurprising that questions regarding evolutionary mechanisms that give rise to cells are sometimes misframed as a forward- engineering problem; that is, How did the cell nd the most efcient way of performing process x? However, there is a fundamental difference between evolution and engineering. Evolution does not always proceed along an optimized path leading to the observed modern state. Viewing each trait as the result of an iterative and mechanistic, rather than teleological, process leading to these solutions changes the way investigations are under- taken and data are interpreted. Although it may remain useful to ask What is the selective advantage of a given trait?, knowing that the evolutionary path is not always direct and constant allows the investigator to consider multiple advantages and possibly entertain alternative explanations beyond selection. Therefore, it may be more productive to answer the how behind evolutionary cell biological questions and to reconstruct the steps and evo- lutionary details for the emergence of a given trait, thus deriving process from the patterns observed across multi- ple examples. Although signicant progress has been made in devel- oping model cell-biological systems across eukaryotes (e.g., Dictyostelium, Toxoplasma, Trypanosoma, Arabidopsis) and analyzing molecular evolution to deduce the origins of protein complexes and their resident organelles, yielding some of the discoveries described above, many areas re- main unexplored. For example, the consequences of popu- lation genetics have not been fully explored in the context of cellular evolution [53]. Similarly, although there have been attempts to correlate geology with cellular evolution [54], particularly regarding the origin of life [55], this aspect is often overlooked by cell biologists. Furthermore, the mechanisms of emergence of evolutionary innovations, such as organelle inheritance, that combine multiple, well adapted cellular components remains to be better eluci- dated [56]. Finally, as our understanding of systems biolo- gy matures and omic data types become increasingly available, we will be able to integrate information about the timing and context of genes and proteins into various models of cellular evolution [57]. With tractable progress being made on concrete mecha- nistic questions, this is truly an exciting time as the biology of the cell can now be parsed in the light of evolution [58]. Acknowledgments The authors thank the members of the Dacks laboratory, as well as W. Ford Doolittle and Holly Goodson, for critical comments on the manuscript and for discussion. J.B.D. and L.D.B. also thank the staff at the Banff Centre for the Arts for their generosity and hospitality during the ooding that occurred in Alberta in June 2013, at which time signicant work on the writing of this manuscript took place. F.D.M. is the recipient of a Vanier Canada Graduate Scholarship from the Canadian Institutes of Health Research (CIHR) and a Full-Time Studentship from Alberta Innovates Health Solutions. L.D.B. was supported by a National Science and Engineering Council of Canada (NSERC) Undergraduate Student Re- search Award. J.B.D. is Canada Research Chair (Tier II) in Evolutionary Cell Biology. Research in the Rachubinski laboratory is supported by grants 9208, 15131, and 53326 fromthe CIHR. 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