Experimental Cell Research 93 (1975) 127-135
ULTRAVIOLET LIGHT MICROBEAM IRRADIATION OF THE MICROTUBULES IN SINGLE HELIOZOAN AXOPODIA C. D. OCKLEFORD’ Department of Zoology, The University, St Andrew, Fife, Scotland, and Institutfiir Biologie der Universitiit, 74, Tiibingen, Auf der Morgenstelle 28, BRD
SUMMARY Irradiation of single axopodia near their distal ends with ultraviolet light induces axopodial retraction which continues for a short period after irradiation has ceased and appears to depend on the reduction in length of a microtubular axoneme. Altering the target site of the microbeam to the mid-point of an axopodium and prolonging the irradiatton time results in the amputation of the distal portion of the axopodium. Both retraction and amputation may involve an ultraviolet light dependent breakdown of cytoplasmic microtubules. Axopodia grow out again after retraction and after amputation. The rates of growth of axopodia shortened in both these ways has been compared. Axopodia which have been caused to retract grow at a mean rate which is faster than that of axopodia which have been amputated.
Slender cell extensions known as axopodia (fig. 1) radiate from the cell bodies of heliozoans [5]. In this organism each axopodium contains a supporting microtubular structure, the axoneme which is based on a modified nuclear envelope [27]. Since the discovery of axonemal microtubules by Kitching [21] many investigators have made use of the rare opportunity afforded by the system for the study of microtubules in a situation of relative isolation from the rest of the cell [l, 23,27,29,40,42]. In his recent review, Bardele [3] concludes that heliozoan axonemal microtubules belong to the labile class designated cytoplasmic microtubules by Behnke & Forer [4]. This class contains the intensive’ Present address: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 IQP, UK. 9-751809
ly studied microtubules of the mitotic apparatus of dividing cells [7, 14,22,24]. Low concentrations of several substances (including mitotic inhibitors) dissolved in heliozoan culture medium cause the breakdown of these sensitive axonemal microtubules and shortening of axopodia [30, 32, 411. Thus there is usually a correlation between the presence of intact microtubules and the formation and maintenance of long thin cellular extensions [42, 441. Although obviously indicated it has not yet been explicitly demonstrated that microtubules support axopodia and that growth of microtubules causes axopodial extension. Consequently there is a need for evidence which will determine whether microtubules are the primary target of treatments which cause retraction of axopodia. Colchicine causes axopodial retraction [26,41] and has also been shown to bind stoichiometrically Exprl Cell Res 93 (1975)
spindle fibre birefringence is caused by the oriented spindle microtubules [ 121. However, in at least one case loss of birefringence has been shown to coincide with spindle microtubule destruction [2]. Experimental irradiation of axopodia with ultraviolet microbeams therefore could help to provide answers to two questions. (I) does ultraviolet microbeam irradiation destroy axonemal microtubules? (2) Is the destruction localised to the area of irradiation as would be expected if its action in this context is primarily on components restricted to axopodia, or does it spread to all other axopodia as would be expected if the destructive effect is mediated by other cellular components? MATERIALS
AND METHODS
The heliozoan Actinophrys sol was maintained in the laboratory as previouslydescribed [27]. Organisms from 14-day-old cultures were isolated using finely were irradiated drawn out glass pipettes. Actinophrys using an ultraviolet microbeam apparatus designed by Starling [36]. A mercury vapour lamp (Zeiss Jena Fig. I. Ac/irwph~.v JO/, living organism. Each fine 220/HB050) illuminated a half-silvered mirror in the inradial projection from the cell body (axopodium) i< termediate tube of a Zeiss WL microscope. The ultrasupported internally by a microtubular axoneme. violet (UV) light was reflected down by the mirror and Microbeams were used to irradiate the tips (sh&~l
ULTRAVIOLET LIGHT MICROBEAM IRRADIATION OF THE MICROTUBULES IN SINGLE HELIOZOAN AXOPODIA C. D. OCKLEFORD’ Department of Zoology, The University, St Andrew, Fife, Scotland, and Institutfiir Biologie der Universitiit, 74, Tiibingen, Auf der Morgenstelle 28, BRD
SUMMARY Irradiation of single axopodia near their distal ends with ultraviolet light induces axopodial retraction which continues for a short period after irradiation has ceased and appears to depend on the reduction in length of a microtubular axoneme. Altering the target site of the microbeam to the mid-point of an axopodium and prolonging the irradiatton time results in the amputation of the distal portion of the axopodium. Both retraction and amputation may involve an ultraviolet light dependent breakdown of cytoplasmic microtubules. Axopodia grow out again after retraction and after amputation. The rates of growth of axopodia shortened in both these ways has been compared. Axopodia which have been caused to retract grow at a mean rate which is faster than that of axopodia which have been amputated.
Slender cell extensions known as axopodia (fig. 1) radiate from the cell bodies of heliozoans [5]. In this organism each axopodium contains a supporting microtubular structure, the axoneme which is based on a modified nuclear envelope [27]. Since the discovery of axonemal microtubules by Kitching [21] many investigators have made use of the rare opportunity afforded by the system for the study of microtubules in a situation of relative isolation from the rest of the cell [l, 23,27,29,40,42]. In his recent review, Bardele [3] concludes that heliozoan axonemal microtubules belong to the labile class designated cytoplasmic microtubules by Behnke & Forer [4]. This class contains the intensive’ Present address: Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 IQP, UK. 9-751809
ly studied microtubules of the mitotic apparatus of dividing cells [7, 14,22,24]. Low concentrations of several substances (including mitotic inhibitors) dissolved in heliozoan culture medium cause the breakdown of these sensitive axonemal microtubules and shortening of axopodia [30, 32, 411. Thus there is usually a correlation between the presence of intact microtubules and the formation and maintenance of long thin cellular extensions [42, 441. Although obviously indicated it has not yet been explicitly demonstrated that microtubules support axopodia and that growth of microtubules causes axopodial extension. Consequently there is a need for evidence which will determine whether microtubules are the primary target of treatments which cause retraction of axopodia. Colchicine causes axopodial retraction [26,41] and has also been shown to bind stoichiometrically Exprl Cell Res 93 (1975)
spindle fibre birefringence is caused by the oriented spindle microtubules [ 121. However, in at least one case loss of birefringence has been shown to coincide with spindle microtubule destruction [2]. Experimental irradiation of axopodia with ultraviolet microbeams therefore could help to provide answers to two questions. (I) does ultraviolet microbeam irradiation destroy axonemal microtubules? (2) Is the destruction localised to the area of irradiation as would be expected if its action in this context is primarily on components restricted to axopodia, or does it spread to all other axopodia as would be expected if the destructive effect is mediated by other cellular components? MATERIALS
AND METHODS
The heliozoan Actinophrys sol was maintained in the laboratory as previouslydescribed [27]. Organisms from 14-day-old cultures were isolated using finely were irradiated drawn out glass pipettes. Actinophrys using an ultraviolet microbeam apparatus designed by Starling [36]. A mercury vapour lamp (Zeiss Jena Fig. I. Ac/irwph~.v JO/, living organism. Each fine 220/HB050) illuminated a half-silvered mirror in the inradial projection from the cell body (axopodium) i< termediate tube of a Zeiss WL microscope. The ultrasupported internally by a microtubular axoneme. violet (UV) light was reflected down by the mirror and Microbeams were used to irradiate the tips (sh&~l
