Specific contact between mating structure membranes observed in conditional fusion-defective Chlamydomonas mutants

Forest, Charlene L.

doi:10.1016/0014-4827(83)90194-5

Cited by 23 publications

(18 citation statements)

References 12 publications

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“…Second, progression from membrane adhesion to complete membrane fusion and coalescence of the two gametes is rapid. Using electron microscopy, we and others have been unable to capture membrane adhesion between mating structures of live wild-type gametes of opposite mating types under standard culture conditions (Goodenough et al, 1982;Forest, 1983;Forest, 1987;Liu et al, 2008). Third, soon after a zygote is formed, its flagella become non-adhesive and therefore the frequency of cell-body contacts between zygotes and non-fused gametes is dramatically reduced.…”

Section: Restricting Gamete Fusion In Chlamydomonasmentioning

confidence: 95%

“…To do this, we generated a minus gamete strain that expressed a wild-type, tagged form of HAP2 and was capable of mating structure adhesion, but was blocked at a separate step in the membrane fusion reaction. As described above, gam-10 gametes had been reported to be defective in fusion at the non-permissive temperature (32°C), but competent for mating structure adhesion as evidenced by the ability of the gam-10 minus gametes to adhere to plus gametes at their mating structures (Forest, 1983). During our earlier studies on mating structure adhesion (Misamore et al, 2003;Liu et al, 2008) we were unable to confirm whether gam-10 gametes were capable of mating structure adhesion or to determine if gam-10 cells possessed a mutant allele of HAP2 because the strain was unavailable to us.…”

Section: Research Articlementioning

confidence: 96%

“…S1 in the supplementary material) by co-transformation with the pSI103 plasmid (Silflow et al, 2001;Schroda et al, 2000;Kindle, 1990;Sizova et al, 2001). The gam-10 minus strain (CC-4164), which has a temperature-sensitive, fusion-defective phenotype (Goodenough et al, 1976;Forest, 1983), recently became available and was obtained from Dr Charlene Forest (Brooklyn, NY, USA). The HAP2-HA plasmid was introduced into the gam-10 strain by co-transformation with the pSI103 plasmid.…”

Section: Cells and Cell Culturementioning

confidence: 99%

“…In wild-type gametes, fusion occurs almost immediately after mating structure adhesion, making it difficult to detect even pairs of cells adhering at their mating structures (Liu et al, 2008). To optimize detection of multiple mating structure adhesions, we mixed wild-type plus gametes and fusion-defective hap2 minus gametes in the same ratios as above, fixed with glutaraldehyde (Forest, 1983), and determined the numbers of cells in clusters of three adhering to each other by their mating structures.…”

Section: Research Articlementioning

confidence: 99%

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Membrane fusion triggers rapid degradation of two gamete-specific, fusion-essential proteins in a membrane block to polygamy inChlamydomonas

Liu

Misamore

Snell³

2010

Development

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SUMMARYThe plasma membranes of gametes are specialized for fusion, yet, once fusion occurs, in many organisms the new zygote becomes incapable of further membrane fusion reactions. The molecular mechanisms that underlie this loss of fusion capacity (block to polygamy) remain unknown. During fertilization in the green alga Chlamydomonas, the plus gamete-specific membrane protein FUS1 is required for adhesion between the apically localized sites on the plasma membranes of plus and minus gametes that are specialized for fusion, and the minus-specific membrane protein HAP2 is essential for completion of the membrane fusion reaction. HAP2 (GCS1) family members are also required for fertilization in Arabidopsis, and for the membrane fusion reaction in the malaria organism Plasmodium berghei. Here, we tested whether Chlamydomonas gamete fusion triggers alterations in FUS1 and HAP2 and renders the plasma membranes of the cells incapable of subsequent fusion. We find that, even though the fusogenic sites support multi-cell adhesions, triploid zygotes are rare, indicating a fusion-triggered block to the membrane fusion reaction. Consistent with the extinction of fusogenic capacity, both FUS1 and HAP2 are degraded upon fusion. The rapid, fusion-triggered cleavage of HAP2 in zygotes is distinct from degradation occurring during constitutive turnover in gametes. Thus, gamete fusion triggers specific degradation of fusion-essential proteins and renders the zygote incapable of fusion. Our results provide the first molecular explanation for a membrane block to polygamy in any organism.

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Section: Restricting Gamete Fusion In Chlamydomonasmentioning

confidence: 95%

Section: Research Articlementioning

confidence: 96%

Section: Cells and Cell Culturementioning

confidence: 99%

Section: Research Articlementioning

confidence: 99%

See 2 more Smart Citations

Membrane fusion triggers rapid degradation of two gamete-specific, fusion-essential proteins in a membrane block to polygamy inChlamydomonas

Liu

Misamore

Snell³

2010

Development

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“…Chlamydomonas reinhardtii, wild type (wt), strain 137c and 3 mt-, fusion-defective mutants, gum-1 [7,8], gum-10 andgam-1 1 [4], isolated from 137c were used for these experiments. Cells were grown with continuous light at 24" C on solidified TAP medium [lo].…”

Section: Materials and Methods Cell Culturementioning

confidence: 99%

Mating Structure Differences Demonstrated by Freeze‐Fracture Analysis of Fusion‐Defective Chlamydomonas Gametes

Forest

Ojakian²

1989

The Journal of Protozoology

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While the mating structure of unmated mating type minus (mt‐) gametes of Chlamydomonas reinhardtii has few intramembrane particles (IMPs), activation results in movement of IMPs to its center. Analysis of freeze‐fractured replicas of wild type (wt) mt‐ and 3 mt‐ fusion‐defective mutants, gam‐1, gam‐10 and gam‐11, before and after activation with wt+ flagella, provides a basis for suggesting that some of the IMPs in mt‐ mating structures, particularly a subset of particles that partitions to the E face, may be fusion‐controlling molecules. Unmated gametes of gam‐10 show a full range of images, from particle‐free to fully activated, with both the P and E face of the mating structure revealing approximately twice as many IMPs as those observed on wt. Unactivated gametes of gam‐1 and gam‐11 appear identical to wt‐. After activation, the mating structures of all of these gametes appear to have approximately the same number of IMPs. If the sizes of particles for these mutants are compared to wild type at the restrictive temperature, all 3 mutants have significantly smaller IMPs on the E face; before mating, in the plasma membrane and after mating, in the mating structure. At 34° C, the gam‐1‐II mating structure appears to be missing most of the particles from 15.5 to 16.5 nm in diameter, while all gametes with the ability to fuse have an equivalent percentage of their mating structure particles in this size range. The possibility that an IMP in this size range represents a protein that may be responsible for gamete fusion is discussed.

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–SH and S–S involvement in Chlamydomonas mating

Forest

1985

Gamete Research

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Reagents that block or cross‐link sulfhydryl (–SH) groups and those that reduce disulfide (S–S) bonds have been tested for their effects on mating in Chlamydomonas reinhardii. Wild‐type (wt) gametes of mating type + (mt+) and mt−, and a fusion‐defective mt− mutant, gam‐11, were studied. Differential sensitivities of mt+ vs mt− and of wt mt− vs gam‐11 mt− were analyzed. Concentrations of reagents that did not disrupt flagellar agglutination, the first stage of the mating reaction, were generally used. Pretreatment of mt+ gametes with the membrane permeable –SH reducing agent dithiothreitol (DTT) inhibits flagellar sexual signaling at concentrations that do not inhibit any part of the mating reaction of mt− gametes. Wt mt− is more sensitive than wt mt+ to inhibition by low concentrations of p‐chloromercuribenzoate sulfonate (pCMBS), an organic mercurial. The membrane‐impermeable reducing agent, reduced glutathione (GSH), also preferentially inhibits wt mt−. Gam‐11 mt−, a fusion‐defective mutant, which has been used to study the sensitivity of the adhesion of the plasma membrane‐associated mating structures, is less sensitive to GSH and pCMBS inhibition that is wt mt−. DDT and pCMBS cause an increase in mating structure adhesion in pretreated gam‐11. The differential inhibition of pair and group formation during gam‐11 × wt mt+ matings has suggested a possible mechanism for mating structure adhesion.

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Specific contact between mating structure membranes observed in conditional fusion-defective Chlamydomonas mutants

Cited by 23 publications

References 12 publications

Membrane fusion triggers rapid degradation of two gamete-specific, fusion-essential proteins in a membrane block to polygamy inChlamydomonas

Membrane fusion triggers rapid degradation of two gamete-specific, fusion-essential proteins in a membrane block to polygamy inChlamydomonas

Mating Structure Differences Demonstrated by Freeze‐Fracture Analysis of Fusion‐Defective Chlamydomonas Gametes

–SH and S–S involvement in Chlamydomonas mating

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