Identification and Characterization of LFD-2, a Predicted Fringe Protein Required for Membrane Integrity during Cell Fusion in Neurospora crassa

Abstract

The molecular mechanisms of membrane merger during somatic cell fusion in eukaryotic species are poorly understood. In the filamentous fungus Neurospora crassa, somatic cell fusion occurs between genetically identical germinated asexual spores (germlings) and between hyphae to form the interconnected network characteristic of a filamentous fungal colony. In N. crassa, two proteins have been identified to function at the step of membrane fusion during somatic cell fusion: PRM1 and LFD-1. The absence of either one of these two proteins results in an increase of germling pairs arrested during cell fusion with tightly appressed plasma membranes and an increase in the frequency of cell lysis of adhered germlings. The level of cell lysis in ⌬Prm1 or ⌬lfd-1 germlings is dependent on the extracellular calcium concentration. An available transcriptional profile data set was used to identify genes encoding predicted transmembrane proteins that showed reduced expression levels in germlings cultured in the absence of extracellular calcium. From these analyses, we identified a mutant (lfd-2, for late fusion defect-2) that showed a calcium-dependent cell lysis phenotype. lfd-2 encodes a protein with a Fringe domain and showed endoplasmic reticulum and Golgi membrane localization. The deletion of an additional gene predicted to encode a low-affinity calcium transporter, fig1, also resulted in a strain that showed a calcium-dependent cell lysis phenotype. Genetic analyses showed that LFD-2 and FIG1 likely function in separate pathways to regulate aspects of membrane merger and repair during cell fusion. Membrane fusion plays an important role in intracellular trafficking of vesicles, exo-and endocytosis, fertilization, the entry of enveloped animal viruses into their host cell, and syncytium formation during developmental processes (1-3). In intracellular vesicle trafficking, SNARE proteins facilitate membrane merger by forming coiled-coil interactions between v-SNARES on vesicles and t-SNARES on target membranes to bring membranes into close proximity (1, 4). The mechanisms that direct cell-cell fusion are less well understood, although genetic screens have identified candidate molecules, termed fusogens, which are membrane-incorporated proteins that facilitate membrane fusion. For example, in Caenorhabditis elegans, two fusogenic glycoproteins, EFF-1 and AFF-1, are necessary and sufficient to fuse cells (5, 6), but their conservation is restricted to nematodes and related organisms (7).In the yeast Saccharomyces cerevisiae, a number of proteins that are involved in mating cell fusion have been identified (8)(9)(10)(11)(12)(13)(14). Mating pairs between strains bearing deletions of Prm1p or Fig1p show tightly appressed plasma membranes, indicating a defect in membrane fusion (10, 12). Mating pairs of prm1⌬ or fig1⌬ strains also show increased cell lysis, the frequency of which is influenced by the concentration of extracellular calcium (12, 15). However, Prm1p and Fig1p are not fusogens, as neither is necessary or sufficient f...

Plasma Membrane Integrity During Cell–Cell Fusion and in Response to Pore-Forming Drugs Is Promoted by the Penta-EF-Hand Protein PEF1 inNeurospora crassa

“…In addition to PRM1, earlier studies identified three additional factors, whose absence results in aberrant fusion and increased lysis rates during germling fusion, LFD-1, LFD-2, and FIG1 (Palma-Guerrero et al 2014, 2015. To test if the observed role of PEF1 is specific for DPrm1-caused defects or if it is of broader significance, we introduced the Dpef1 gene knockout into the Dlfd-1, Dlfd-2 and Dfig1 mutants and compared the lysis rates of the single and the respective double mutants on standard and Ca 2+ -reduced growth medium.…”

“…They appear to ensure fusion fidelity, probably by organizing or spatially restricting the fusion machinery (Fleissner et al 2009;Palma-Guerrero et al 2014). The lack of LFD-2 or FIG1 also causes increased lysis rates, but no adhering membranes are observed, indicating distinct functions from PRM1 and LFD-1 (Palma-Guerrero et al 2015). Interestingly, the lysis rates in Dfig1 do not further increase in the absence of pef1.…”

Identification of Glutaminyl Cyclase Genes Involved in Pyroglutamate Modification of Fungal Lignocellulolytic Enzymes

“…(A) Fluorescence microscopy of Δqc - 1 Δqc - 2 strain expressing either qc - 1 - sfGFP or qc - 2 - sfGFP . (B) Fluorescence microscopy of heterokaryotic cells expressing qc - 1 - sfGFP and sec61 - mCherry (53). Bars, 10 μm.…”

“…Homokaryotic transformants were obtained as described above. The Sec61::mCherry strain was described previously (53). …”

“…Confocal microscopy was performed as described previously (53) using a Leica SD6000 microscope with a 100× 1.4-numerical-aperture (NA) oil immersion objective equipped with a Yokogawa CSU-X1 spinning disc head and a 488-nm or 561-nm laser controlled by MetaMorph software. ImageJ software was used for false color and overlaying images.…”