Nuclear movement in filamentous fungi

One of the most striking features of eukaryotic cells is the organization of specific functions into organelles such as nuclei, mitochondria, chloroplasts, the endoplasmic reticulum, vacuoles, peroxisomes or the Golgi apparatus. These membrane-surrounded compartments are not synthesized de novo but are bequeathed to daughter cells during cell division. The successful transmittance of organelles to daughter cells requires the growth, division and separation of these compartments and involves a complex machinery consisting of cytoskeletal components, mechanochemical motor proteins and regulatory factors. Organelles such as nuclei, which are present in most cells in a single copy, must be precisely positioned prior to cytokinesis. In many eukaryotic cells the cleavage plane for cell division is defined by the location of the nucleus prior to mitosis. Nuclear positioning is thus absolutely crucial in the unequal cell divisions that occur during development and embryogenesis. Yeast and filamentous fungi are excellent organisms for the molecular analysis of nuclear migration because of their amenability to a broad variety of powerful analytical methods unavailable in higher eukaryotes. Filamentous fungi are especially attractive models because the longitudinally elongated cells grow by apical tip extension and the organelles are often required to migrate long distances. This review describes nuclear migration in filamentous fungi, the approaches used for and the results of its molecular analysis and the projection of the results to other organisms.     

Cell Biology of Prokaryotic Organelles

Mounting evidence in recent years has challenged the dogma that prokaryotes are simple and undefined cells devoid of an organized subcellular architecture. In fact, proteins once thought to be the purely eukaryotic inventions, including relatives of actin and tubulin control prokaryotic cell shape, DNA segregation, and cytokinesis. Similarly, compartmentalization, commonly noted as a distinguishing feature of eukaryotic cells, is also prevalent in the prokaryotic world in the form of protein-bounded and lipid-bounded organelles. In this article we highlight some of these prokaryotic organelles and discuss the current knowledge on their ultrastructure and the molecular mechanisms of their biogenesis and maintenance.The emergence of eukaryotes in a world dominated by prokaryotes is one of the defining moments in the evolution of modern day organisms. Although it is clear that the central metabolic and information processing machineries of eukaryotes and prokaryotes share a common ancestry, the origins of the complex eukaryotic cell plan remain mysterious. Eukaryotic cells are typified by the presence of intracellular organelles that compartmentalize essential biochemical reactions whereas their prokaryotic counterparts generally lack such sophisticated subspecialization of the cytoplasmic space. In most cases, this textbook categorization of eukaryotes and prokaryotes holds true. However, decades of research have shown that a number of unique and diverse organelles can be found in the prokaryotic world raising the possibility that the ability to form organelles may have existed before the divergence of eukaryotes from prokaryotes (Shively 2006).Skeptical readers might wonder if a prokaryotic structure can really be defined as an organelle. Here we categorize any compartment bounded by a biological membrane with a dedicated biochemical function as an organelle. This simple and broad definition presents cells, be they eukaryotes or prokaryotes, with a similar set of challenges that need to be addressed to successfully build an intracellular compartment. First, an organism needs to mold a cellular membrane into a desired shape and size. Next, the compartment must be populated with the proper set of proteins that carry out the activity of the organelle. Finally, the cell must ensure the proper localization, maintenance and segregation of these compartments across the cell cycle. Eukaryotic cells perform these difficult mechanistic steps using dedicated molecular pathways. Thus, if connections exist between prokaryotic and eukaryotic organelles it seems likely that relatives of these molecules may be involved in the biogenesis and maintenance of prokaryotic organelles as well.Prokaryotic organelles can be generally divided into two major groups based on the composition of the membrane layer surrounding them. First are the cellular structures bounded by a nonunit membrane such a protein shell or a lipid monolayer (Shively 2006). Well-known examples of these compartments include lipid bodies, polyhydroxy butyrate granules, carboxysomes, and gas vacuoles. The second class consists of those organelles that are surrounded by a lipid-bilayer membrane, an arrangement that is reminiscent of the canonical organelles of the eukaryotic endomembrane system. Therefore, this article is dedicated to a detailed exploration of three prokaryotic lipid-bilayer bounded organelle systems: the magnetosomes of magnetotactic bacteria, photosynthetic membranes, and the internal membrane structures of the Planctomycetes. In each case, we present the most recent findings on the ultrastructure of these organelles and highlight the molecular mechanisms that control their formation, dynamics, and segregation. We also highlight some protein-bounded compartments to present the reader with a more complete view of prokaryotic compartmentalization.     

Mapping the subcellular protein distribution in three human cell lines

The subcellular locations of proteins are closely related to their function and constitute an essential aspect for understanding the complex machinery of living cells. A systematic effort has been initiated to map the protein distribution in three functionally different cell lines with the aim to provide a subcellular localization index for at least one representative protein from all human protein-encoding genes. Here, we present the results of more than 3500 proteins mapped to 16 subcellular compartments. The results indicate a ubiquitous protein expression with a majority of the proteins found in all three cell lines and a large portion localized to two or more compartments. The inter-relationships between the subcellular compartments are visualized in a protein-compartment network based on all detected proteins. Hierarchical clustering was performed to determine how closely related the organelles are in terms of protein constituents and compare the proteins detected in each cell type. Our results show distinct organelle proteomes, well conserved across the cell types, and demonstrate that biochemically similar organelles are grouped together.     

Cytoarchitectural reorganization of rabbit colonic goblet cells during baseline secretion

Light and electron microscopy were coupled with point counting methods to quantitate shape and volume changes of goblet cells during their migration and maturation from the base of the crypt to the colonic surface epithelium in the rabbit. After differentiation, goblet cells attain a broad pyramidal configuration in the basal third of the crypt. The cells elongate and dramatically decrease in volume as they move into the surface epithelium. The distributions and volume fractions of organelles were found to vary considerably, depending on the location of the goblet cell in the epithelium. Mucin granules are initially synthesized throughout the cytoplasm, but become increasingly concentrated as the cell matures. Organelles involved in synthesis such as the Golgi apparatus and rough endoplasmic reticulum (RER) similarly attain a more concentrated arrangement as the cell moves up in the crypt. The mean cell volume decreases from 1,228.8 microns3 for cells in the basal third of the crypt to 541.3 microns3 for goblet cells on the surface. Most organelles decrease in proportion to this decrease, although a disproportionately large decrease in the RER was measured. When actual subcellular volumes are calculated, a net decrease in several subcellular compartments is detected. This loss of granules and organelles is accomplished by the continual synthesis and secretion of mucin granules. Cytoplasm and organelles become entrapped in the upward movement of granules towards the cell apex, become irretrievably isolated, and are sloughed into the crypt lumen. This process accounts for the decrease in cell volume and contributes to the altered cytoarchitecture of the cell.     

Characterization of SNAREs determines the absence of a typical Golgi apparatus in the ancient eukaryote Giardia lamblia

Giardia is a eukaryotic protozoal parasite with unusual characteristics, such as the absence of a morphologically evident Golgi apparatus. Although both constitutive and regulated pathways for protein secretion are evident in Giardia, little is known about the mechanisms involved in vesicular docking and fusion. In higher eukaryotes, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) of the vesicle-associated membrane protein and syntaxin families play essential roles in these processes. In this work we identified and characterized genes for 17 SNAREs in Giardia to define the minimal set of subcellular organelles present during growth and encystation, in particular the presence or not of a Golgi apparatus. Expression and localization of all Giardia SNAREs demonstrate their presence in distinct subcellular compartments, which may represent the extent of the endomembrane system in eukaryotes. Remarkably, Giardia SNAREs, homologous to Golgi SNAREs from other organisms, do not allow the detection of a typical Golgi apparatus in either proliferating or differentiating trophozoites. However, some features of the Golgi, such as the packaging and sorting function, seem to be performed by the endoplasmic reticulum and/or the nuclear envelope. Moreover, depletion of individual genes demonstrated that several SNAREs are essential for viability, whereas others are dispensable. Thus, Giardia requires a smaller number of SNAREs compared with other eukaryotes to accomplish all of the vesicle trafficking events that are critical for the growth and differentiation of this important human pathogen.     

Hydrogenosomes and Mitosomes: Conservation and Evolution of Functions1

ABSTRACT. The field studying unusual mitochondria in microbial eukaryotes has come full circle. Some 10–15 years ago it had the evangelical task of informing the wider scientific community that not all eukaryotes had mitochondria. Advances in the field indicated that although some protists might not have mitochondria, the presence of genes of mitochondrial ancestry suggested their lineage once had. The subsequent discovery of mitochondrial compartments in all supposedly amitochondriate protists studied so far indicates that all eukaryotes do have mitochondria indeed. This assertion has fuelled novel eukaryotic origin theories and weakened others. But what do we know about these unusual mitochondria from anaerobic protists? Have they all converged onto similar roles? Iron–sulphur cluster assembly is often hailed as the unifying feature of these organelles. However, the iron–sulphur protein that is so important that a complete organelle is being maintained has not been identified. Is it to be expected that all unusual mitochondria perform the same physiological role? These organelles have been found in numerous protists occupying different ecological niches. Different selection pressures operate on different organisms so there is no reason to suspect that their mitochondria should all be the same.     

Comparative analysis of an experimental subcellular protein localization assay and in silico prediction methods

The subcellular localization of a protein can provide important information about its function within the cell. As eukaryotic cells and particularly mammalian cells are characterized by a high degree of compartmentalization, most protein activities can be assigned to particular cellular compartments. The categorization of proteins by their subcellular localization is therefore one of the essential goals of the functional annotation of the human genome. We previously performed a subcellular localization screen of 52 proteins encoded on human chromosome 21. In the current study, we compared the experimental localization data to the in silico results generated by nine leading software packages with different prediction resolutions. The comparison revealed striking differences between the programs in the accuracy of their subcellular protein localization predictions. Our results strongly suggest that the recently developed predictors utilizing multiple prediction methods tend to provide significantly better performance over purely sequence-based or homology-based predictions.     

Subcellular compartments and protein topogenesis

A cell is surrounded by a plasma membrane. It contains various organelles, most of which are enclosed by limiting membranes. The intracellular space is thus divided into a number of subcellular compartments. Structurally, a cell is composed of membranes and the spaces enclosed by those membranes. In order to classify these compartments, the extracellular space has been designated S1 and whenever a unit membrane structure is crossed to arrive at the next space, one is added to term; the cytoplasmic space becomes S2, the intraluminal space of the endoplasmic reticulum and the intermembrane space of the mitochondria S3, and the matrix space of the mitochondria S4. Similarly, the plasma membrane is M1, the outer membrane of the mitochondria M2, and the inner counterpart M3. This classification of the subcellular compartments is useful in understanding a number of complicated cellular structures and functions. The intracellular transport of newly synthesized protein (protein topogenesis) and the probable development of subcellular organelles during phylogenesis of eukaryotic cells is discussed in terms of these subcellular compartments.     

Mitochondria and hydrogenosomes are two forms of the same fundamental organelle

Published data suggest that hydrogenosomes, organelles found in diverse anaerobic eukaryotes that make energy and hydrogen, were once mitochondria. As hydrogenosomes generally lack a genome, the conversion is probably one way. The sources of the key hydrogenosomal enzymes, pyruvate : ferredoxin oxidoreductase (PFO) and hydrogenase, are not resolved by current phylogenetic analyses, but it is likely that both were present at an early stage of eukaryotic evolution. Once thought to be restricted to a few unusual anaerobic eukaryotes, the proteins are intimately integrated into the fabric of diverse eukaryotic cells, where they are targeted to different cell compartments, and not just hydrogenosomes. There is no evidence supporting the view that PFO and hydrogenase originated from the mitochondrial endosymbiont, as posited by the hydrogen hypothesis for eukaryogenesis. Other organelles derived from mitochondria have now been described in anaerobic and parasitic microbial eukaryotes, including species that were once thought to have diverged before the mitochondrial symbiosis. It thus seems possible that all eukaryotes may eventually be shown to contain an organelle of mitochondrial ancestry, to which different types of biochemistry can be targeted. It remains to be seen if, despite their obvious differences, this family of organelles shares a common function of importance for the eukaryotic cell, other than energy production, that might provide the underlying selection pressure for organelle retention.     

Characterisation of organellar proteomes: a guide to subcellular proteomic fractionation and analysis

Subcellular fractionation is being widely used to increase our understanding of the proteome. Fractionation is often coupled with 2-DE, thus allowing the visualisation of proteins and their subsequent identification and characterisation by MS. Whilst this strategy should be effective, to date, there has been little or no consideration given to differences in the mass, pI, hydropathy or abundance of proteins in the organelles and how analytical strategies can be tailored to match the idiosyncrasies of proteins in each particular compartment. To address this, we analysed 3962 Saccharomyces cerevisiae proteins, previously localised to one or more of 22 subcellular compartments. Different compartments showed significantly different distributions of protein pI and hydropathy. Mitochondrial and ER proteins showed the most dramatic differences to other organelles, in their protein pIs and hydropathy, respectively. We show that organelles can be clustered by similarities in these physicochemical protein characteristics. Interestingly, the distribution of protein abundance was also significantly different between many organelles. Our results show that to fully explore subcellular fractions of the proteome, specific analytical strategies should be employed. We outline strategies for all 22 subcellular compartments.     

The unique features of starch metabolism in red algae.

Red algae (Rhodophyceae) are photosynthetic eukaryotes that accumulate starch granules outside of their plastids. The starch granules from red algae (floridean starch) show structural similarities with higher plant starch granules but lack amylose. Recent studies have indicated that the extra-plastidic starch synthesis in red algae proceeds via a UDP glucose-selective alpha-glucan synthase, in analogy with the cytosolic pathway of glycogen synthesis in other eukaryotes. On the other hand, plastidic starch synthesis in green cells occurs selectively via ADP glucose in analogy with the pathway of glycogen synthesis in prokaryotes from which plastids have evolved. Given the emerging consensus of a monophyletic origin of plastids, it would appear that the capacity for starch synthesis selectively evolved from the alpha-glucan synthesizing machinery of the host ancestor and its endosymbiont in red algae and green algae, respectively. This implies the evolution of fundamentally different functional relationships between the different subcellular compartments with regard to photosynthetic carbon metabolism in these organisms. It is suggested that the biochemical and molecular elucidation of floridean starch synthesis may offer new insights into the metabolic strategies of photosynthetic eukaryotes.     

Enzymes of the heme biosynthetic pathway in the nonphotosynthetic alga Polytomella sp

Heme biosynthesis involves a number of enzymatic steps which in eukaryotes take place in different cell compartments. Enzyme compartmentalization differs between photosynthetic and nonphotosynthetic eukaryotes. Here we investigated the structures and subcellular localizations of three enzymes involved in the heme pathway in Polytomella sp., a colorless alga evolutionarily related to the green alga Chlamydomonas reinhardtii. Functional complementation of Escherichia coli mutant strains was used to isolate cDNAs encoding three heme biosynthetic enzymes, glutamate-1-semialdehyde aminotransferase, protoporphyrinogen IX oxidase, and ferrochelatase. All three proteins show highest similarity to their counterparts in photosynthetic organisms, including C. reinhardtii. All three proteins have N-terminal extensions suggestive of intracellular targeting, and immunoblot studies indicate their enrichment in a dense cell fraction that is enriched in amyloplasts. These results suggest that even though the plastids of Polytomella sp. are not photosynthetically active, they are the major site of heme biosynthesis. The presence of a gene for glutamate-1-semialdehyde aminotransferase suggests that Polytomella sp. uses the five-carbon pathway for synthesis of the heme precursor 5-aminolevulinic acid.     

The pgp1 mutant locus of Arabidopsis encodes a phosphatidylglycerolphosphate synthase with impaired activity

Phosphatidylglycerol is a ubiquitous phospholipid that is also present in the photosynthetic membranes of plants. Multiple independent lines of evidence suggest that this lipid plays a critical role for the proper function of photosynthetic membranes and cold acclimation. In eukaryotes, different subcellular compartments are competent for the biosynthesis of phosphatidylglycerol. Details on the plant-specific pathways in different organelles are scarce. Here, we describe a phosphatidylglycerol biosynthesis-deficient mutant of Arabidopsis, pgp1. The overall content of phosphatidylglycerol is reduced by 30%. This mutant carries a point mutation in the CDP-alcohol phosphotransferase motif of the phosphatidylglycerolphosphate synthase (EC 2.7.8.5) isoform encoded by a gene on chromosome 2. The mutant shows an 80% reduction in plastidic phosphatidylglycerolphosphate synthase activity consistent with the plastidic location of this particular isoform. Mutant plants are pale green, and their photosynthesis is impaired. This mutant provides a promising new tool to elucidate the biosynthesis and function of plastidic phosphatidylglycerol in seed plants.     

Degenerate mitochondria

Mitochondria are the main sites of biological energy generation in eukaryotes. These organelles are remnants of a bacterial endosymbiont that took up residence inside a host cell over 1,500 million years ago. Comparative genomics studies suggest that the mitochondrion is monophyletic in origin. Thus, the original mitochondrial endosymbiont has evolved independently in anaerobic and aerobic environments that are inhabited by diverse eukaryotic lineages. This process has resulted in a collection of morphologically, genetically and functionally heterogeneous organelle variants that include anaerobic and aerobic mitochondria, hydrogenosomes and mitosomes. Current studies aim to determine whether a central common function drives the retention of mitochondrial organelles in different eukaryotic organisms.     

SMP domain proteins in membrane lipid dynamics

Synaptotagmin-like mitochondrial-lipid-binding (SMP) domain proteins are evolutionarily conserved family of proteins in eukaryotes that localize between the endoplasmic reticulum (ER) and either the plasma membrane (PM) or other organelles. They are involved in tethering of these membrane contact sites through interaction with other proteins and membrane lipids. Recent structural and biochemical studies have demonstrated that SMP domain proteins transport a wide variety of lipid species by the ability of the SMP domain to harbor lipids through its unique hydrophobic cavity. Growing evidence suggests that SMP domain proteins play critical roles in cell physiology by their actions at membrane contact sites. In this review, we summarize the functions of SMP domain proteins and their direct roles in lipid transport across different membrane compartments. We also discuss their physiological functions in organisms as well as “bypass” pathways that act in parallel with SMP domain proteins at membrane contact sites.     

Isotope labelling of Rubisco subunits provides in vivo information on subcellular biosynthesis and exchange of amino acids between compartments

The architecture of plant metabolism includes substantial duplication of metabolite pools and enzyme catalyzed reactions in different subcellular compartments. This poses challenges for understanding the regulation of metabolism particularly in primary metabolism and amino acid biosynthesis. To explore the extent to which amino acids are made in single compartments and to gain insight into the metabolic precursors from which they derive, we used steady state (13) C labelling and analysed labelling in protein amino acids from plastid and cytosol. Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is a major component of green tissues and its large and small subunits are synthesized from different pools of amino acids in the plastid and cytosol, respectively. Developing Brassica napus embryos were cultured in the presence of [U-(13) C]-sucrose, [U-(13) C]-glucose, [U-(13) C]-glutamine or [U-(13) C]-alanine to generate proteins. The large subunits (LSU) and small subunits (SSU) of Rubisco were isolated and the labelling in their constituent amino acids was analysed by gas chromatography-mass spectrometry. Amino acids including alanine, glycine and serine exhibited different (13) C enrichment in the LSU and SSU, demonstrating that these pools have different metabolic origins and are not isotopically equilibrated between the plastid and cytosol on the time scale of cellular growth. Potential extensions of this novel approach to other macromolecules, organelles and cell types of eukaryotes are discussed.     

Membraneless organelles: P granules in Caenorhabditis elegans

Membraneless organelles are distinct compartments within a cell that are not enclosed by a traditional lipid membrane and instead form through a process called liquid‐liquid phase separation. Examples of these non‐membrane‐bound organelles include nucleoli, stress granules, P bodies, pericentriolar material and germ granules. Many recent studies have used Caenorhabditis elegans germ granules, known as P granules, to expand our understanding of the formation of these unique cellular compartments. From this work, we know that proteins with intrinsically disordered regions (IDRs) play a critical role in the process of phase separation. IDR phase separation is further tuned through their interactions with RNA and through protein modifications such as phosphorylation and methylation. These findings from C elegans, combined with work done in other model organisms, continue to provide insight into the formation of membraneless organelles and the important role they play in compartmentalizing cellular processes.