Arginine-Enriched Mixed-Charge Domains Provide Cohesion for Nuclear Speckle Condensation.

Low-complexity protein domains promote the formation of various biomolecular condensates. However, in many cases, the precise sequence features governing condensate formation and identity remain unclear. Here, we investigate the role of intrinsically disordered mixed-charge domains (MCDs) in nuclear speckle condensation. Proteins composed exclusively of arginine-aspartic acid dipeptide repeats undergo length-dependent condensation and speckle incorporation. Substituting arginine with lysine in synthetic and natural speckle-associated MCDs abolishes these activities, identifying a key role for multivalent contacts through arginine's guanidinium ion. MCDs can synergize with a speckle-associated RNA recognition motif to promote speckle specificity and residence. MCD behavior is tunable through net-charge: increasing negative charge abolishes condensation and speckle incorporation. Contrastingly, increasing positive charge through arginine leads to enhanced condensation, speckle enlargement, decreased splicing factor mobility, and defective mRNA export. Together, these results identify key sequence determinants of MCD-promoted speckle condensation and link the dynamic material properties of speckles with function in mRNA processing.

Evolutionary novelty in gravity sensing through horizontal gene transfer and high-order protein assembly.

Horizontal gene transfer (HGT) can promote evolutionary adaptation by transforming a species' relationship to the environment. In most well-understood cases of HGT, acquired and donor functions appear to remain closely related. Thus, the degree to which HGT can lead to evolutionary novelties remains unclear. Mucorales fungi sense gravity through the sedimentation of vacuolar protein crystals. Here, we identify the octahedral crystal matrix protein (OCTIN). Phylogenetic analysis strongly supports acquisition of octin by HGT from bacteria. A bacterial OCTIN forms high-order periplasmic oligomers, and inter-molecular disulphide bonds are formed by both fungal and bacterial OCTINs, suggesting that they share elements of a conserved assembly mechanism. However, estimated sedimentation velocities preclude a gravity-sensing function for the bacterial structures. Together, our data suggest that HGT from bacteria into the Mucorales allowed a dramatic increase in assembly scale and emergence of the gravity-sensing function. We conclude that HGT can lead to evolutionary novelties that emerge depending on the physiological and cellular context of protein assembly.

Artificial import substrates reveal an omnivorous peroxisomal importomer.

The peroxisome matrix protein importomer has the remarkable ability to transport oligomeric protein substrates across the bilayer. However, the selectivity and relation between import and overall peroxisome homeostasis remain unclear. Here, we microinject artificial import substrates and employ quantitative microscopy to probe limits and capabilities of the importomer. DNA and polysaccharides are "piggyback" imported when noncovalently bound by a peroxisome targeting signal (PTS)-bearing protein. A dimerization domain that can be tuned to systematically vary the binding dissociation constant (Kd ) shows that a Kd in the millimolar range is sufficient to promote piggyback import. Microinjection of import substrate at high levels results in peroxisome growth and a proportional accumulation of peroxisome membrane proteins (PMPs). However, corresponding PMP mRNAs do not accumulate, suggesting that this response is posttranscriptionally regulated. Together, our data show that the importomer can tolerate diverse macromolecular species. Coupling between matrix import and membrane biogenesis suggests that matrix protein expression levels can be sufficient to regulate peroxisome size.

Predicting the targeting of tail-anchored proteins to subcellular compartments in mammalian cells.

Tail-anchored (TA) proteins contain a single transmembrane domain (TMD) at the C-terminus that anchors them to the membranes of organelles where they mediate critical cellular processes. Accordingly, mutations in genes encoding TA proteins have been identified in a number of severe inherited disorders. Despite the importance of correctly targeting a TA protein to its appropriate membrane, the mechanisms and signals involved are not fully understood. In this study, we identify additional peroxisomal TA proteins, discover more proteins that are present on multiple organelles, and reveal that a combination of TMD hydrophobicity and tail charge determines targeting to distinct organelle locations in mammals. Specifically, an increase in tail charge can override a hydrophobic TMD signal and re-direct a protein from the ER to peroxisomes or mitochondria and vice versa. We show that subtle changes in those parameters can shift TA proteins between organelles, explaining why peroxisomes and mitochondria have many of the same TA proteins. This enabled us to associate characteristic physicochemical parameters in TA proteins with particular organelle groups. Using this classification allowed successful prediction of the location of uncharacterized TA proteins for the first time.

Peroxisome assembly and functional diversity in eukaryotic microorganisms.

Peroxisomes are core eukaryotic organelles that generally function in lipid metabolism and detoxification of reactive oxygen species, but they are increasingly associated with taxa-specific metabolic, cellular, and developmental functions. Here, we present a brief overview of peroxisome assembly, followed by a discussion of their functional diversification. Matrix protein import occurs through a remarkable translocon that can accommodate folded and even oligomeric proteins. Metabolically specialized peroxisomes include glycosomes of trypanosomes, which have come to compartmentalize most of the glycolytic pathway and play a role in developmental signal transduction. The differentiation of physically distinct subcompartments also contributes to peroxisome diversification; in the clade of filamentous ascomycetes, dense-core Woronin bodies bud from peroxisomes to gate cell-to-cell channels. Here, the import of oligomeric cargo is central to the mechanism of subcompartment specification. In general, the acquisition of a tripeptide peroxisome targeting signal by nonperoxisomal proteins appears to be a recurrent step in the evolution of peroxisome diversity.

Intrinsically disordered proteins aggregate at fungal cell-to-cell channels and regulate intercellular connectivity.

Like animals and plants, multicellular fungi possess cell-to-cell channels (septal pores) that allow intercellular communication and transport. Here, using a combination of MS of Woronin body-associated proteins and a bioinformatics approach that identifies related proteins based on composition and character, we identify 17 septal pore-associated (SPA) proteins that localize to the septal pore in rings and pore-centered foci. SPA proteins are not homologous at the primary sequence level but share overall physical properties with intrinsically disordered proteins. Some SPA proteins form aggregates at the septal pore, and in vitro assembly assays suggest aggregation through a nonamyloidal mechanism involving mainly α-helical and disordered structures. SPA loss-of-function phenotypes include excessive septation, septal pore degeneration, and uncontrolled Woronin body activation. Together, our data identify the septal pore as a complex subcellular compartment and focal point for the assembly of unstructured proteins controlling diverse aspects of intercellular connectivity.

Multiple modes for gatekeeping at fungal cell-to-cell channels.

Cell-to-cell channels appear to be indispensable for successful multicellular organization and arose independently in animals, plants and fungi. Most of the fungi obtain nutrients from the environment by growing in an exploratory and invasive manner, and this ability depends on multicellular filaments known as hyphae. These cells grow by tip extension and can be divided into compartments by cell walls that typically retain a central pore that allows intercellular transport and cooperation. In the major clade of filamentous Ascomycota, integrity of this coenocytic organization is maintained by Woronin body organelles, which function as emergency patches of septal pores. In this issue of Molecular Microbiology, Bleichrodt and co-workers show that Woronin bodies can also form tight reversible associations with the pore and further link this to variation in levels of compartmental gene expression. These data define an additional modality of Woronin body-dependent gatekeeping. This commentary focuses on the implications of this work and the potential role of different modes of pore gating in controlling the growth and development of fungal tissues.

Cooperative motility, force generation and mechanosensing in a foraging non-photosynthetic diatom.

Diatoms are ancestrally photosynthetic microalgae. However, some underwent a major evolutionary transition, losing photosynthesis to become obligate heterotrophs. The molecular and physiological basis for this transition is unclear. Here, we isolate and characterize new strains of non-photosynthetic diatoms from the coastal waters of Singapore. These diatoms occupy diverse ecological niches and display glucose-mediated catabolite repression, a classical feature of bacterial and fungal heterotrophs. Live-cell imaging reveals deposition of secreted extracellular polymeric substance (EPS). Diatoms moving on pre-existing EPS trails (runners) move faster than those laying new trails (blazers). This leads to cell-to-cell coupling where runners can push blazers to make them move faster. Calibrated micropipettes measure substantial single-cell pushing forces, which are consistent with high-order myosin motor cooperativity. Collisions that impede forward motion induce reversal, revealing navigation-related force sensing. Together, these data identify aspects of metabolism and motility that are likely to promote and underpin diatom heterotrophy.

A tether for Woronin body inheritance is associated with evolutionary variation in organelle positioning.

Eukaryotic organelles evolve to support the lifestyle of evolutionarily related organisms. In the fungi, filamentous Ascomycetes possess dense-core organelles called Woronin bodies (WBs). These organelles originate from peroxisomes and perform an adaptive function to seal septal pores in response to cellular wounding. Here, we identify Leashin, an organellar tether required for WB inheritance, and associate it with evolutionary variation in the subcellular pattern of WB distribution. In Neurospora, the leashin (lah) locus encodes two related adjacent genes. N-terminal sequences of LAH-1 bind WBs via the WB-specific membrane protein WSC, and C-terminal sequences are required for WB inheritance by cell cortex association. LAH-2 is localized to the hyphal apex and septal pore rim and plays a role in colonial growth. In most species, WBs are tethered directly to the pore rim, however, Neurospora and relatives have evolved a delocalized pattern of cortex association. Using a new method for the construction of chromosomally encoded fusion proteins, marker fusion tagging (MFT), we show that a LAH-1/LAH-2 fusion can reproduce the ancestral pattern in Neurospora. Our results identify the link between the WB and cell cortex and suggest that splitting of leashin played a key role in the adaptive evolution of organelle localization.

Marker fusion tagging, a new method for production of chromosomally encoded fusion proteins.

A new gene-tagging method (marker fusion tagging [MFT]) is demonstrated for Neurospora crassa and Magnaporthe oryzae. Translational fusions between the hygromycin B resistance gene and various markers are inserted into genes of interest by homologous recombination to produce chromosomally encoded fusion proteins. This method can produce tags at any position and create deletion alleles that maintain N- and C-terminal sequences. We show the utility of MFT by producing enhanced green fluorescent protein (EGFP) tags in proteins localized to nuclei, spindle pole bodies, septal pore plugs, Woronin bodies, developing septa, and the endoplasmic reticulum.

Fungal Wound Healing through Instantaneous Protoplasmic Gelation.

Multicellular organisms employ fluid transport networks to overcome the limit of diffusion and promote essential long-distance transport. Connectivity and pressurization render these networks especially vulnerable to wounding. To mitigate this risk, animals, plants, and multicellular fungi independently evolved elaborate clotting and plugging mechanisms. In the septate filamentous fungi, membrane-bound organelles plug septal pores in wounded hyphae. By contrast, vegetative hyphae in the early-diverging Mucoromycota are largely aseptate, and how their hyphae respond to wounding is unknown. Here, we show that wounding in the Mucorales leads to explosive protoplasmic discharge that is rapidly terminated by protoplasmic gelation. We identify Mucoromycota-specific Gellin proteins, whose loss of function leads to uncontrolled wound-induced protoplasmic bleeding. Gellins contain ten related ß-trefoil Gll domains, each of which possesses unique features that impart distinct gelation-related properties: some readily unfold and form high-order sheet-like structures when subjected to mechanical force from flow, while others possess hydrophobic motifs that enable membrane binding. In cell-free reconstitution, sheet-like structures formed by a partial Gellin incorporate membranous organelles. Together, these data define a mechanistic basis for regulated protoplasmic gelation, and provide new design principles for the development of artificial flow-responsive biomaterials.

Spitzenkörper assembly mechanisms reveal conserved features of fungal and metazoan polarity scaffolds.

The Spitzenkörper (SPK) constitutes a collection of secretory vesicles and polarity-related proteins intimately associated with polarized growth of fungal hyphae. Many SPK-localized proteins are known, but their assembly and dynamics remain poorly understood. Here, we identify protein-protein interaction cascades leading to assembly of two SPK scaffolds and recruitment of diverse effectors in Neurospora crassa. Both scaffolds are transported to the SPK by the myosin V motor (MYO-5), with the coiled-coil protein SPZ-1 acting as cargo adaptor. Neither scaffold appears to be required for accumulation of SPK secretory vesicles. One scaffold consists of Leashin-2 (LAH-2), which is required for SPK localization of the signalling kinase COT-1 and the glycolysis enzyme GPI-1. The other scaffold comprises a complex of Janus-1 (JNS-1) and the polarisome protein SPA-2. Via its Spa homology domain (SHD), SPA-2 recruits a calponin domain-containing F-actin effector (CCP-1). The SHD NMR structure reveals a conserved surface groove required for effector binding. Similarities between SPA-2/JNS-1 and the metazoan GIT/PIX complex identify foundational features of the cell polarity apparatus that predate the fungal-metazoan divergence.

Ypt31/32 GTPases and their novel F-box effector protein Rcy1 regulate protein recycling.

Ypt/Rab GTPases control various aspects of vesicle formation and targeting via their diverse effectors. We report a new role for these GTPases in protein recycling through a novel effector. The F-box protein Rcy1, which mediates plasma membrane recycling, is identified here as a downstream effector of the Ypt31/32 GTPase pair because it binds active GTP-bound Ypt31/32 and colocalizes with these GTPases on late Golgi and endosomes. Furthermore, Ypt31/32 regulates the polarized localization and half-life of Rcy1. This suggests that Ypt/Rabs can regulate the protein level of their effectors, in addition to the established ways by which they control their effectors. We show that like Rcy1, Ypt31/32 regulate the coupled phosphorylation and recycling of the plasma membrane v-SNARE Snc1. Moreover, Ypt31/32 and Rcy1 regulate the recycling of the furin-homolog Kex2 to the Golgi. Therefore, Ypt31/32 and Rcy1 mediate endosome-to-Golgi transport, because this is the only step shared by Snc1 and Kex2. Finally, we show that Rcy1 physically interacts with Snc1. Based on this result and because F-box proteins serve as adaptors between specific substrates and ubiquitin ligases, we propose that Ypt31/32 GTPases regulate the function of Rcy1 in the phosphorylation and/or ubiquitination of proteins that recycle through the Golgi.

Polarized gene expression determines woronin body formation at the leading edge of the fungal colony.

The Woronin body (WB) is a peroxisome-related organelle that is centered on a crystalline core of the HEX-1 protein, which functions to seal septal pores of filamentous ascomycetes in response to cellular damage. Here, we investigate the cellular and genetic control of WB-formation and show that polarized hex-1 gene expression determines WB-biogenesis at the growing hyphal apex. We find that intron splicing is coupled to efficient hex-1 gene expression and strikingly, when the yellow fluorescent protein was expressed from hex-1 regulatory sequences, we observed a fluorescent gradient that was maximal in apical cells. Moreover, endogenous hex-1 transcripts were specifically enriched at the leading edge of the fungal colony, whereas other transcripts accumulated in basal regions. Time-lapse confocal microscopy showed that HEX-1 crystals normally formed in the vicinity of the hyphal apex in large peroxisomes, which matured and were immobilized at the cell periphery as cells underwent septation. When the hex-1 structural gene was expressed from regulatory sequences of an abundant, basally localized transcript, WB-core formation was redetermined to basal regions of the colony, and these strains displayed loss-of-function phenotypes specifically in apical hyphal compartments. These results show that apically localized gene expression is a key determinant of spatially restricted WB-assembly. We suggest that this type of regulation may be widely used to determine cellular activity in apical regions of the fungal hypha.

Innovation and constraint leading to complex multicellularity in the Ascomycota.

The advent of complex multicellularity (CM) was a pivotal event in the evolution of animals, plants and fungi. In the fungal Ascomycota, CM is based on hyphal filaments and arose in the Pezizomycotina. The genus Neolecta defines an enigma: phylogenetically placed in a related group containing mostly yeasts, Neolecta nevertheless possesses Pezizomycotina-like CM. Here we sequence the Neolecta irregularis genome and identify CM-associated functions by searching for genes conserved in Neolecta and the Pezizomycotina, which are absent or divergent in budding or fission yeasts. This group of 1,050 genes is enriched for functions related to diverse endomembrane systems and their organization. Remarkably, most show evidence for divergence in both yeasts. Using functional genomics, we identify new genes involved in fungal complexification. Together, these data show that rudimentary multicellularity is deeply rooted in the Ascomycota. Extensive parallel gene divergence during simplification and constraint leading to CM suggest a deterministic process where shared modes of cellular organization select for similarly configured organelle- and transport-related machineries.

Cellular Subcompartments through Cytoplasmic Streaming.

Cytoplasmic streaming occurs in diverse cell types, where it generally serves a transport function. Here, we examine streaming in multicellular fungal hyphae and identify an additional function wherein regimented streaming forms distinct cytoplasmic subcompartments. In the hypha, cytoplasm flows directionally from cell to cell through septal pores. Using live-cell imaging and computer simulations, we identify a flow pattern that produces vortices (eddies) on the upstream side of the septum. Nuclei can be immobilized in these microfluidic eddies, where they form multinucleate aggregates and accumulate foci of the HDA-2 histone deacetylase-associated factor, SPA-19. Pores experiencing flow degenerate in the absence of SPA-19, suggesting that eddy-trapped nuclei function to reinforce the septum. Together, our data show that eddies comprise a subcellular niche favoring nuclear differentiation and that subcompartments can be self-organized as a consequence of regimented cytoplasmic streaming.

Mapping Woronin body position in Aspergillus nidulans.

The positions of all Woronin bodies in five germlings of Aspergillus nidulans prepared by plunge freezing and freeze substitution were determined by transmission electron microscopy. As expected, Woronin bodies were found near septa. High numbers of morphologically identical organelles were also found in apical regions. To verify that these organelles were authentic Woronin bodies, we used antibodies raised against the Neurospora crassa Woronin body matrix protein Hex1. Anti-Hex1 antibodies labeled Woronin bodies at septa and in apical regions of A. nidulans. In germlings that had not yet formed septa, at least fifty percent of Woronin bodies were found within 2.5 µm of the tip. In germ tubes that had formed septa, the total number of Woronin bodies remained the same, but only twenty percent were near the tip. Our results clearly establish that Woronin bodies are found in apical regions of Aspergillus germ tubes and suggest that Woronin bodies are transported from the apex to the more basal regions of the cell immediately before or during septation.

Hydrophobic handoff for direct delivery of peroxisome tail-anchored proteins.

Tail-anchored (TA) proteins are inserted into membranes post-translationally through a C-terminal transmembrane domain (TMD). The PEX19 protein binds peroxisome TA proteins in the cytoplasm and delivers them to the membrane through the PEX3 receptor protein. An amphipathic segment in PEX19 promotes docking on PEX3. However, how this leads to substrate insertion is unknown. Here we reconstitute peroxisome TA protein biogenesis into two sequential steps of substrate TMD engagement and membrane insertion. We identify a series of previously uncharacterized amphipathic segments in PEX19 and identify one whose hydrophobicity is required for membrane insertion, but not TMD chaperone activity or PEX3 binding. A membrane-proximal hydrophobic surface of PEX3 promotes an unconventional form of membrane intercalation, and is also required for TMD insertion. Together, these data support a mechanism in which hydrophobic moieties in the TMD chaperone and its membrane-associated receptor act in a concerted manner to prompt TMD release and membrane insertion.

Fungal evo-devo: organelles and multicellular complexity.

Peroxisome-derived Woronin bodies of the Ascomycota phyla, and the endoplasmic reticulum (ER)-derived septal pore cap (SPC) of the Basidiomycota, are both fungal organelles that prevent cytoplasmic bleeding when multicellular hyphal filaments are wounded. Analysis of Woronin body constituent proteins suggests that these organelles evolved in part through gene duplication and co-opting of non-essential genes for new functions, indicating that new organelles can arise through typical evolutionary mechanisms. Interestingly, clades possessing the Woronin body and SPC also produce the largest and most complex multicellular fungal reproductive structures. Certain Woronin body and SPC mutants have defects in growth and development, suggesting functions beyond cellular wound healing. I argue that studying these specialized systems will help to reveal the basis for fungal diversity and provide general principles for co-evolution of organelles and multicellular complexity.

Import oligomers induce positive feedback to promote peroxisome differentiation and control organelle abundance.

A fundamental question in cell biology is how cells control organelle composition and abundance. Woronin bodies are fungal peroxisomes centered on a crystalline core of the self-assembled HEX protein. Despite using the canonical peroxisome import machinery for biogenesis, Woronin bodies are scarce compared to the overall peroxisome population. Here, we show that HEX oligomers promote the differentiation of a subpopulation of peroxisomes, which become enlarged and highly active in matrix protein import. HEX physically associates with the essential matrix import peroxin, PEX26, and promotes its enrichment in the membrane of differentiated peroxisomes. In addition, a PEX26 mutant that disrupts differentiation produces increased numbers of aberrantly small Woronin bodies. Our data suggest a mechanism where HEX oligomers recruit a key component of the import machinery, which promotes the import of additional HEX. This type of positive feedback provides a basic mechanism for the production of an organelle subpopulation of distinct composition and abundance.