Microcompartmentation of glycolytic enzymes in cultured cells.

The microcompartmentation of aldolase and glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) was investigated in four different cell types (3T3 cells, SV 40 transformed 3T3 cells, mouse fibroblasts, chick embryo cardiomyocytes) combining cell permeabilization and indirect immunofluorescence technique. Permeabilization of the cells prior to fixation released the soluble fractions, whilst the total amount of enzymes was preserved in nonpermeabilized cells. Both enzymes exist in a soluble as well as in a structure-bound form. The soluble fraction of aldolase and GAPDH is distributed homogeneously throughout the cytoplasm, excluding the nucleus and vesicles. The permeabilization-resistant form is associated with the actin cytoskeleton. A considerable amount of both enzymes is located in the perinuclear region and cannot be attributed to a definite structure. Comparing the staining patterns of aldolase and GAPDH in four different cell types we found that the distribution of the enzymes corresponds with diverse forms of actin cytoskeletal organization of these cells. The codistribution is maintained in cells treated with cytochalasin D.

Quantitation of the volume of liquid injected into cells by means of pressure.

The amount of TRITC-labeled bovine serum albumin (TRITC-BSA) released from the tip of standardized microcapillaries at different injection pressures and times was investigated. Three test systems were used: (a) formation of droplets of the test solution (TRITC-BSA) in paraffin oil, (b) injection of the test solution into buffer droplets suspended in paraffin oil, and (c) injection into living 3T3 cells. The amount of test substance released was determined by scanning fluorometry. The first procedure (a) allows the fluorometrically determined amount of TRITC-BSA to be related to the volume of released test solution. For this rather large pressures (about 700 hPa) are required to overcome the surface forces counteracting droplet formation. The volume of the spheres was evaluated from photomicrographs of the droplets. The values obtained correlate very well with those determined by measuring the fluorescence emitted by the droplets. Injection into preformed droplets of buffer (b) can be performed with pressures in the range used for injecting cells (50-400 hPa). High reproducibility in the volume released is obtained with a single microcapillary; however, large variations exist between different capillaries, although these should theoretically be of equal diameter. The volume injected into living cells (c) under a given condition may vary by a factor of 5 or more. This variation may be due to viscosity differences of cytoplasm. We recommend, therefore, injection of fluorescent marker substances together with the test substance, enabling the injected volume to be determined by scanning fluorometry or by image analysis.