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Scholarly Works (5 results)

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UC Berkeley Electronic Theses and Dissertations (2019)

All multicellular organisms face a challenge: resources must be distributed throughout their bodies to ensure continued growth and coordinate cellular activities, but once resources are socialized, how can cells be coerced into working for the organism as a whole, rather than individually reproducing at the expense of the body they inhabit? Solutions to this freeloader problem usually rely on aligning the reproductive interests of all cells in a body by ensuring all are genetically identical. However, such solutions pose problems for organisms with indeterminate colonial growth habits, a lifestyle characterized by vegetative expansion, fragmentation, and reintegration of separated fragments. The filamentous fungus Neurospora crassa exemplifies this lifestyle. Its body is a cross-linked syncytial network produced by the fusion of many cells. Individual nuclei flow throughout the colony, making all the products of each nucleus potentially available to freeloaders. To keep the reproductive interests of all nuclei aligned, N. crassa must restrict fusion to genetically cells. The fungus prevents non-self fusion using an array of non-self recognition (NSR) systems.

NSR systems are encoded by polymorphic kind recognition loci. Each NSR locus enables cells to behave differently toward other individuals with the same haplotype than they do toward individuals with distinct haplotypes. At least two haplotypes at an NSR locus must exist in a population for the system to function. If many NSR loci are spread throughout an organisms genome, the net output of all NSR systems transitions to kin recognition; this allows an organism to identify genetically identical and distinct individuals with high fidelity. N. crassa encodes 15 confirmed NSR loci, with dozens more suspected based on sequencing and phenotypic investigations. Different NSR systems function at various stages in N. crassa’s life cycle, and during distinct phases of sociality. The first phase of sociality is called communication, and occurs when two cells detect each other and reorient their growth to intersect. The Determinant Of Communication (DOC) NSR system restricts communication between non-identical cells before they touch.

We published our discovery of the DOC system in 2016. At the time, we knew haplotypes at the doc locus were necessary and sufficient to specify which cells will be recognized as self. We also reported that DOC-2 localized to the cellular periphery and DOC-1 oscillated between the cell bodies and growing tips of cells along with the MAK-2 complex during intercellular communication. We found the DOC system was not required for self-communication, implying it suppresses communication until a compatible communication group (CG) signal is received. Finally, we identified five distinct communication group haplotypes (CGHs) at the doc locus in an N. crassa population from Louisiana; doc alleles from different CGHs were less than 50% identical at the nucleotide level, and exhibited trans-species polymorphism among N. crassa and other members of its genus.

Little else was certain regarding the DOC system. With neither characterized homologues nor identifiable functional domains, the DOC proteins remained particularly opaque. The primary goal of my doctoral work was to advance our understanding of the DOC system. I attempted to improve our model of DOC-mediated long-distance NSR by investigating how different doc genes and alleles interact in vivo, and evaluating the roles played by various regions of the DOC proteins in regulating communication. In pursuit of these aims, I spent considerable time and effort developing improved methods for quantifying intercellular communication.

After developing and validating a flow cytometry-based communication assay and analysis pipeline, I used this assay to evaluate the communication behavior of N. crassa strains expressing incompatible alleles of the doc genes, multiple incompatible DOC systems, truncated and otherwise manipulated alleles of the doc genes, and CGH1/CGH3 chimeric alleles of doc-1. The results of these experiments support the following model for DOC-mediated long-distance NSR:

As cells begin to communicate, general and CG-specific signals must be exchanged. CG signals are validated through interactions between the middle region of DOC-1 and DOC-2: matched CGH-variants of the DOC proteins are generally required for proper CG specificity. If the DOC system does not receive a compatible CG signal, DOC-1 and DOC-2 both prevent reinforcement of MAK-2 complex oscillations and suppress communication. This suppression involves the actions of the middle and C-terminal regions of DOC-1, and does not require the N-terminal region of DOC-2. If a compatible CG signal is detected, the DOC system allows MAK-2 complex oscillations and chemotropic growth to continue. DOC- 1’s N-terminal region is required to properly derepress communication, and is probably involved in validating CG signals.

    Cover page: Long-Distance Non-Self Recognition and Control of Intercellular Communication in Neurospora crassa
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2020)
    Social cooperation impacts the development and survival of species. In higher taxa, kin recognition occurs via visual, chemical, or tactile cues that dictate cooperative versus competitive interactions. In microbes, the outcome of cooperative versus competitive interactions is conferred by identity at allorecognition loci, so-called kind recognition. In syncytial filamentous fungi, the acquisition of multicellularity is associated with somatic cell fusion within and between colonies. However, such intraspecific cooperation entails risks, as fusion can transmit deleterious genotypes or infectious components that reduce fitness, or give rise to cheaters that can exploit communal goods without contributing to their production. Allorecognition mechanisms in syncytial fungi regulate somatic cell fusion by operating precontact during chemotropic interactions, during cell adherence, and postfusion by triggering programmed cell death reactions. Alleles at fungal allorecognition loci are highly polymorphic, fall into distinct haplogroups, and show evolutionary signatures of balancing selection, similar to allorecognition loci across the tree of life.
      Cover page: Conflict, Competition, and Cooperation Regulate Social Interactions in Filamentous Fungi
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2016)
      Microorganisms are capable of communication and cooperation to perform social activities. Cooperation can be enforced using kind discrimination mechanisms in which individuals preferentially help or punish others, depending on genetic relatedness only at certain loci. In the filamentous fungus Neurospora crassa, genetically identical asexual spores (germlings) communicate and fuse in a highly regulated process, which is associated with fitness benefits during colony establishment. Recognition and chemotropic interactions between isogenic germlings requires oscillation of the mitogen-activated protein kinase (MAPK) signal transduction protein complex (NRC-1, MEK-2, MAK-2, and the scaffold protein HAM-5) to specialized cell fusion structures termed conidial anastomosis tubes. Using a population of 110 wild N. crassa isolates, we investigated germling fusion between genetically unrelated individuals and discovered that chemotropic interactions are regulated by kind discrimination. Distinct communication groups were identified, in which germlings within one communication group interacted at high frequency, while germlings from different communication groups avoided each other. Bulk segregant analysis followed by whole genome resequencing identified three linked genes (doc-1, doc-2, and doc-3), which were associated with communication group phenotype. Alleles at doc-1, doc-2, and doc-3 fell into five haplotypes that showed transspecies polymorphism. Swapping doc-1 and doc-2 alleles from different communication group strains was necessary and sufficient to confer communication group affiliation. During chemotropic interactions, DOC-1 oscillated with MAK-2 to the tips of conidial anastomosis tubes, while DOC-2 was statically localized to the plasma membrane. Our data indicate that doc-1, doc-2, and doc-3 function as "greenbeard" genes, involved in mediating long-distance kind recognition that involves actively searching for one's own type, resulting in cooperation between non-genealogical relatives. Our findings serve as a basis for investigations into the mechanisms associated with attraction, fusion, and kind recognition in other eukaryotic species.
        Cover page: Characterization of Greenbeard Genes Involved in Long-Distance Kind Discrimination in a Microbial Eukaryote
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2019)
        Somatic cell fusion and conspecific cooperation are crucial social traits for microbial unicellular-to-multicellular transitions, colony expansion, and substrate foraging but are also associated with risks of parasitism. We identified a cell wall remodeling (cwr) checkpoint that acts upon cell contact to assess genetic compatibility and regulate cell wall dissolution during somatic cell fusion in a wild population of the filamentous fungus Neurospora crassa. Non-allelic interactions between two linked loci, cwr-1 and cwr-2, were necessary and sufficient to block cell fusion: cwr-1 encodes a polysaccharide monooxygenase (PMO), a class of enzymes associated with extracellular degradative capacities, and cwr-2 encodes a predicted transmembrane protein. Mutations of sites in CWR-1 essential for PMO catalytic activity abolished the block in cell fusion between formerly incompatible strains. In Neurospora, alleles cwr-1 and cwr-2 were highly polymorphic, fell into distinct haplogroups, and showed trans-species polymorphisms. Distinct haplogroups and trans-species polymorphisms at cwr-1 and cwr-2 were also identified in the distantly related genus Fusarium, suggesting convergent evolution. Proteins involved in chemotropic processes showed extended localization at contact sites, suggesting that cwr regulates the transition between chemotropic growth and cell wall dissolution. Our work revealed an allorecognition surveillance system based on kind discrimination that inhibits cooperative behavior in fungi by blocking cell fusion upon contact, contributing to fungal immunity by preventing formation of chimeras between genetically non-identical colonies.
          Cover page: Allorecognition upon Fungal Cell-Cell Contact Determines Social Cooperation and Impacts the Acquisition of Multicellularity
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2014)
          Previous observations made by our laboratory indicate that Bruton's tyrosine kinase (Btk) may play an important role in the pathophysiology of local inflammation in acute lung injury (ALI)/acute respiratory distress syndrome (ARDS). We have shown that there is cross talk between FcγRIIa and TLR4 in alveolar neutrophils from patients with ALI/ARDS and that Btk mediates the molecular cooperation between these two receptors. To study the function of Btk in vivo we have developed a unique two-hit model of ALI: LPS/immune complex (IC)-induced ALI. Furthermore, we conjugated F(ab)2 fragments of anti-neutrophil antibodies (Ly6G1A8) with specific siRNA for Btk to silence Btk specifically in alveolar neutrophils. It should be stressed that we are the first group to perform noninvasive transfections of neutrophils, both in vitro and in vivo. Importantly, our present findings indicate that silencing Btk in alveolar neutrophils has a dramatic protective effect in mice with LPS/IC-induced ALI, and that Btk regulates neutrophil survival and clearance of apoptotic neutrophils in this model. In conclusion, we put forward a hypothesis that Btk-targeted neutrophil specific therapy is a valid goal of research geared toward restoring homeostasis in lungs of patients with ALI/ARDS.
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