Caffeine-Induced Surface Blebbing and Budding in the Acellular Slime Mold Physarum polycephalum

Abstract

The mechanism of plasma membrane proliferation was studied in the acellular slime mold Physarum polycephalum with the aid of light and electron microscopical techniques. Treatment of protoplasmic drops with a Tris-buffered 15 mᴍ caffeine solution causes surface blebbing and budding over periods of 5-90 min. The process of surface blebbing is coupled to a 5-10-fold increase of the surface area in conjunction with characteristic changes in cytoplasmic morphol­ogy. Successive constriction of blebs exhibiting different sizes and degree of hyalo-granuloptasmic separation leads to the formation of numerous spherical caffeine droplets. During the process of surface budding and droplet formation the total surface area of the original (genuine) protoplasmic drop is not reduced, but continues to grow. Freeze-etch studies show that caffeine concomitantly causes characteristic changes in the fine structure of the plasma membrane. During the initial phase of surface blebbing the original density of intramembranous particles (IMP) is reduced from 3676/μm2 to 1669/μm2 and the PF:EF ratio (IMP/μm2 protoplasmic face: exoplasmic face) shifts from 2.4:1 to 2.8:1. When surface budding is completed the IMP-density in the plasma membrane of single caffeine droplets increases again to 2289/μm2 and the PF:EF ratio changes to 1.5:1. Simultaneously, the isolated caffeine droplets produce numerous small hyaline membrane protrusions, which are pinched off and contain no IMP. Control experiments demonstrate that Tris-buffer without caffeine also shows a weak capacity to induce surface blebbing, to change the IMP-density and the PF:EF ratio (2443/μm2; 1.5:1); but Tris-buffer fails to cause surface budding. On the other hand, different concentrations of sucrose (25-200 mᴍ) can supress to a certain degree both caffeine- and Tris-buffer-induced surface blebbing, but not caffeine-dependent surface budding. The caffeine-effect is reversible insofar as protoplasmic drops with blebbing and budding activity recover to normal morphology, fine structure and locomotion when transferred to physiological conditions. The mechanisms of successive changes in plasma membrane morphology as well as the mode of a participation of the actomyosin system in cell surface dynamics are discussed.