Oscillatory recruitment of signaling proteins to cell tips promotes coordinated behavior during cell fusion

Abstract

Cell-cell communication is essential for coordinating physiological responses in multicellular organisms and is required for various developmental processes, including cell migration, differentiation, and fusion. To facilitate communication, functional differences are usually required between interacting cells, which can be established either genetically or developmentally. However, genetically identical cells in the same developmental state are also capable of communicating, but must avoid self-stimulation. We hypothesized that such cells must alternate their physiological state between signal sending and receiving to allow recognition and behavioral changes. To test this hypothesis, we studied cell communication in the filamentous fungus Neurospora crassa, a simple and experimentally amenable model system. In N. crassa, germinating asexual spores (germlings) of identical genotype chemotropically sense others in close proximity, show attraction-mediated directed growth, and ultimately undergo cell fusion. Here, we report that two proteins required for cell fusion, a MAP kinase (MAK-2) and a protein of unknown molecular function (SO), exhibit rapid oscillatory recruitment to the plasma membranes of interacting germlings undergoing chemotropic interactions via directed growth. Using an inhibitable MAK-2 variant, we show that MAK-2 kinase activity is required both for chemotropic interactions and for oscillation of MAK-2 and SO to opposing cell tips. Thus, N. crassa germlings undergoing chemotropic interactions rapidly alternate between two different physiological states, associated with signal delivery and response. Such spatiotemporal coordination of signaling allows genetically identical and developmentally equivalent cells to avoid self-stimulation and to coordinate their behavior to achieve the beneficial physiological outcome of cell fusion.

Fig. 1.

MAK-2 localizes to CAT tips involved in chemotropic interactions. (A) Wild-type N. crassa conidial germling pairs of identical genotype undergoing chemotropic attraction. (B) Attachment and fusion of the tips (arrow) shown in panel A (20 min later). (Scale bar, 10 μm.) (C) Localization of MAK-2-GFP to the CAT tip (arrow) in a germling (his-3::Pccg1 mak-2-gfp Δmak-2) undergoing chemotropic attraction. (Scale bar, 2 μm.) (D) Western blot showing full length MAK-2-GFP protein (≈67 kDa) produced in the his-3::mak-2-gfp; Δmak-2 (AF-P611) and his-3::Pccg1 mak-2-gfp; Δmak-2 (AF-M512) strains. Strain A9 (his-3::Pccg1 gfp) shows the approximately 26-kDa GFP protein. (E) Complementation of macroscopic phenotype of the Δmak-2 mutant with Pccg1 mak-2-gfp allele.

Fig. 2.

SO and MAK-2 oscillate to opposing CAT tips during chemotropic interactions. (A) Time course of MAK-2-GFP localization in his-3::Pccg1 mak-2-gfp Δmak-2 germling pairs. Graphic representation of MAK-2-GFP localization to CAT tips of two cells undergoing chemotropic attraction. T1 = CAT tip one, T2 = CAT tip two. y axis shows the ratio of relative fluorescence intensity (R.F.I.) in the interacting zone as compared to background. x axis shows time (min). (B) Time course of SO-GFP localization to CATs in his-3::Pccg1 so-gfp Δso germling fusion pairs. Graphic representation of SO-GFP localization to CAT tips of fusion pairs over time. T1 = CAT tip one, T2 = CAT tip two. y axis and x axis identical to (A). (C) Time course of MAK-2-GFP and dsRED-SO localization in germling pairs [(his-3::Pccg1 mak-2-gfp; Δmak-2) + (his-3::Pccg1 dsRED-so Δso)]. Graph shows coordinated oscillation of MAK-2-GFP and dsRED-SO localization in one CAT tip of a germling pair. Fig. S2 shows MAK-2-GFP and dsRED-SO oscillation in both CAT tips. y axis and x axis identical to (A). (Scale bar, 2 μm.)

Fig. 3.

MAK-2 and SO localization during germling fusion. (A) MAK-2-GFP and H1-dsRED colocalize in nuclei of a germling pair [(his-3::Pccg1 mak-2-gfp; Δmak-2) + his-3::H1-dsRED)] during chemotropic interactions (arrow). (B) SO is excluded from nuclei in a germling pair [(his-3::Pccg1 so-gfp Δso) + his-3::H1-dsRED)]; arrow marks a nucleus. Asterisk shows region of chemotropic attraction. (C) MAK-2-GFP localized to CATs tips is not bounded by membrane. his-3::Pccg1 mak-2-gfp; Δmak-2 strain labeled with membrane-selective dye FM4–64 (red). The MAK-2-GFP particles (green; arrow) are either associated with the plasma membrane or free within the cytoplasm. Other organelles are membrane-bound (asterisk). (D) A his-3::Pccg1 so-gfp Δso strain labeled with FM4–64 shows localization of SO-GFP to CAT tips (green; arrows), but not colocalization with membrane bound structures (red). (E) MAK-2-GFP and dsRED-S) localization at cell fusion point after contact between with germlings [(his-3::Pccg1 mak-2-gfp; Δmak-2) + (his-3::Pccg1 dsRED-so Δso)]. dsRED-SO delocalizes from the fusion point after contact. (F) MAK-2-GFP localization around the fusion pore as it enlarges. Z-stack of optical sections of the cells shown in 3E. The axis of stack rotation (relative to Fig. 3E) is marked by an arrow. dsRED-SO is sequestered within vacuoles passing through the fusion pore. (Scale bar, 2 μm.)

Fig. 4.

Inhibition of MAK-2 kinase activity disrupts both MAK-2 and SO oscillation during chemotropic interactions. (A) Inhibition of MAK-2 kinase function by addition of 1NM-PP1 to (his-3::Pccg1 mak-2^Q100G; Δmak-2) germlings causes cessation of MAK-2-GFP oscillation in a partner cell (his-3::Pccg1 mak-2-gfp; Δmak-2). 1NM-PP1 added after 0 min time point. Note lack of MAK-2 oscillation in graphic representation of MAK-2-GFP localization to CAT tip over time. y axis shows the ratio of relative fluorescence intensity (R.F.I.) in the interacting zone as compared to background; x axis shows time. (B) Inhibition of MAK-2 kinase function in one cell (his-3::Pccg1 mak-2^Q100G; Δmak-2) causes a cessation of SO-GFP oscillation in the CAT of a partner cell (his-3::Pccg1 so-gfp Δso). 1NM-PP1 added after 0 min time point. Note lack of SO oscillation in graphic representation of SO-GFP localization in CAT tips after addition of 1NM-PP1. x axis and y axis as in (A). (C) Inhibition of MAK-2 kinase activity in [his-3::Pccg1 mak-2Q100G; Δmak-2 (so-gfp)] germling pairs results in accumulation of the particulate SO-GFP complexes in both cells, which gradually lose polarity and spread around the cell cortex. 1NM-PP1 added after the 0 min time point. Graphic representation of SO-GFP localization to both CAT tips after the addition of 1NM-PP1. Y and x axis are as in (A). (Scale bar, 2 μm.)

Fig. 5.

Model for chemotropic interactions during germling fusion involving MAK-2 and SO functions. Early in chemotropic interactions during germling fusion, MAK-2 and SO show opposing localization around CAT tips. SO is associated with the release of a pulse of diffusible signal, which results in recruitment and activation of MAK-2 at the cell cortex in the partner cell. Kinase-active MAK-2 results in activation of downstream targets, such as components of the cytoskeleton, which are involved in directed growth. MAK-2 kinase activity is required for delocalization of SO from the cell cortex. Inactivation of MAK-2 (by phosphatases or indirect action of SO) results in delocalization from the cell cortex and SO accumulation at the cell cortex. As oscillation continues, both MAK-2 and SO are localized in tight foci associated with future contact sites and fusion pore position.