OCTAHEDRAL CRYSTALS IN PHYCOMYCES. II

Abstract

Phycomyces blakesleeanus sporangiophores contain octahedral crystals throughout their cytoplasm and vacuole . More octahedral crystals were found in the wild-type strain G5 (+) than in the ß-carotene-deficient mutant C5 (-), and much more than in the mutant C141 (-), which is sensitive to only high light intensity . In the wild type, the number of crystals per sporangiophore increased until the sporangiophore reached stage IV, and then decreased . Stage I contained the most crystals per unit volume . Cultures grown in darkness had the maximum number of crystals . Under high light intensity, there was an overall reduction of crystals . The crystals are regular octahedrons . The crystals were isolated from the sporangiophores by a method of sucrose density-gradient centrifugation, They contain nearly 95 % protein, are stable in organic solvents, but can be solubilized in buffer solution above pH 9 .5 and below 2 .5 . The crystals weakly fluoresce with an emission peak at 540 nm, which is affected by irradiation with white light. Absorption spectra of freshly prepared crystals show absorption maxima around 265-285 nm, 350-380 nm, and 450-470 nm . These absorption peaks for the crystals are close to those of the phototropic and lightgrowth action spectra . These data suggest that the crystals may contain a flavoprotein which may be the photoreceptor pigment of Phycomyces. INTRODUCTIONThe fungus Phycomyces blakesleeanus sporangiophores are large single cells, centimeters in length, which are sensitive to light and to other physical stimuli (Shropshire, 1963 ;Bergman et al ., 1969) . Because of their sensitivity to light, Phycomyces is of considerable interest as a model cell for photosensory studies . To date, no photoreceptor structure has been identified, and it is not known what pigment or pigment system is responsible for these light responses . Action spectra for Phycomyces phototropism and light-growth response show absorption around 280, 370-385, 445-455, and 470-485 nm (Curry and Gruen, 1959 ;Delbrück and Shropshire, 1960) .Crystals in Phycomyces sporangiophores were first observed by van Tieghem (1875) . More recently, they have been investigated by Thornton and Thimann (1964), Thornton (1969), Wolken (1969 . However, the function of these crystals in Phycomyces is not known . A hypothesis which we have pursued is that the crystals which are located in the growth zone of the sporangiophores are connected in some way with the photoreceptor process . Microspectrophotometry of these crystals in situ suggested that these crystals might contain a flavine (Wolken, 1969(Wolken, , 1972 .In the following experiments we set out to establish the culture conditions which produced the maximum yield of crystals, to determine where in the cell the crystals are located, and then to isolate the crystals from the sporangiophores . The isolated crystals' chemical nature and their 2 7 8

Optospectroscopic Detection of Primary Reactions Associated with the Graviperception of Phycomyces. Effects of Micro- and Hypergravity

“…These are: (1) bending force (flexure) of the sporangiophore (Dennison, 1961), (2) the sedimentation of statoliths (Schimek et al, 1999a(Schimek et al, , 1999b, and (3) the buoyance of apical lipid globules (Schimek et al, 1999a;Grolig et al, 2003). The statoliths of P. blakesleeanus are identical with the previously described vacuolar protein crystals that display a paracrystalline structure (Ootaki and Wolken, 1973). The protein crystals are made up of of three proteins that contain noncovalently bound pterin-and flavin-like pigments Fries et al, 2002).…”

Statoliths in Phycomyces: Characterization of Octahedral Protein Crystals

“…The method employed for crystal isolation was a modification of the method by Ootaki and Wolken (1973). Stage 1 sporangiophores (without sporangium) were collected and were cut with scissors into small pieces approximately 1 mm long.…”

“…In previous studies we showed that paracrystalline protein bodies, so-called octahedral crystals, occurring in sporangiophores of Phycomyces blakesleeanus function as statoliths (Horie et al, 1998a;Schimek et al, 1999a,b). These octahedral crystals had been known before, but had never been associated with gravitropism (Thornton, 1968(Thornton, , 1969Thornton and Thimann, 1964;Wolken, 1969Wolken, , 1970Ootaki and Wolken, 1973), even though their purported high specific mass (Zalokar, 1969) would have let them appear ideal contenders as statoliths. Octahedral crystals occur in the central vacuoles of the sporangiophore, in which they can move freely.…”

Phycomyces: Electrical Response to Light Stimuli

“…Orientation of the photopigment molecule is related to the early receptor potential observed in photoreceptors and plants (2,9). Experiments have shown that rod crystals, having a vertical orientation, can be observed in Phycomyces sporangiophore under polarized light (7, 10). These crystals appear to be aligned in the growth zone, but whether they are located in or near the membrane has not been determined.…”

“…Sensitivity of the sporangiophore to a variety of physical stimuli has been documented, and the most extensively studied response is the phototropic response (3). To date no specific photoreceptor structure has been identified and it is still inconclusive whether the photoreceptor molecule is a flavin or carotenoid (7,10). Detection of electrical signals from the sporangiophore in response to light excitation has not been reported despite efforts to obtain such data.…”