Acidification of endocytic vesicles has been implicated as a necessary step in various processes including receptor recycling, virus penetration, and the entry of diphtheria toxin into cells . However, there have been few accurate pH measurements in morphologically and biochemically defined endocytic compartments . In this paper, we show that prelysosomal endocytic vesicles in HepG2 human hepatoma cells have an internal pH of approximately 5.4 . (We previously reported that similar vesicles in mouse fibroblasts have a pH of 5.0 .) The pH values were obtained from the fluorescence excitation profile after internalization of fluorescein labeled asialo-orosomucoid (ASOR) . To make fluorescence measurements against the high autofluorescence background, we developed digital image analysis methods for estimating the pH within individual endocytic vesicles or lysosomes . Ultrastructural localization with colloidal gold ASOR demonstrated that the pH measurements were made when ligand was in tubulovesicular structures lacking acid phosphatase activity . Biochemical studies with "'I-ASOR demonstrated that acidification precedes degradation by more than 30 min at 37°C . At 23°C ligand degradation ceases almost entirely, but endocytic vesicle acidification and receptor recycling continue . These results demonstrate that acidification of endocytic vesicles, which causes ligand dissociation, occurs without fusion of endocytic vesicles with lysosomes. Methylamine and monensin raise the pH of endocytic vesicles and cause a ligand-independent loss of receptors . The effects on endocytic vesicle pH are rapidly reversible upon removal of the perturbant, but the effects on cell surface receptors are slowly reversible with methylamine and essentially irreversible with monensin . This suggests that monensin can block receptor recycling at a highly sensitive step beyond the acidification of endocytic vesicles . Taken together with other direct and indirect estimates of endocytic vesicle pH, these studies indicate that endocytic vesicles in many cell types rapidly acidify below pH 5 .5, a pH sufficiently acidic to allow receptor-ligand dissociation and the penetration of some toxin chains and enveloped virus nucleocapsids into the cytoplasm.We recently demonstrated that endocytic vesicles in mouse fibroblasts become acidified shortly after formation (31), and suggested that this early acidification of endocytic vesicles may be a necessary step in processes such as receptor recycling and the penetration of enveloped viruses and diphtheria toxin into the cytoplasm . While there have been several recent studies that support this hypothesis through indirect evidence, there have been very few direct measurements of the pH of endocytic vesicles. In addition to our measurement in mouse fibroblasts using fluorescein-labeled a 2 -macroglobulin as a pH probe, van Renswoude et al . (34) have shown that fluoresceinlabeled transferrin taken up by erythroleukemic cells is in nonlysomal intracellular compartments with an average pH of 5 .5 ...

The microinjection of calcium-saturated calmodulin into living fibroblasts causes the rapid disruption of microtubules and stress fibers in a sharply delimited region concentric with the injection site . This effect is specific to the calcium-bearing form of calmodulin; neither calcium-free calmodulin nor calcium ion at similar levels affects the cytoskeleton . If cells have previously been microinjected with calcium-free calmodulin, elevation of their intracellular calcium levels to 25 mM potentiates the disruption of microtubules throughout the cytoplasm. Approximately 400 mM free calcium is required to cause an equivalent disruption in uninjected cells. The level of calmodulin necessary to disrupt the full complement of cellular microtubules is found to be approximately in 2 :1 molar ratio to tubulin dimer. These results indicate that calmodulin can be localized within the cytoplasm in a calcium-dependent manner and that it can act to regulate the calcium lability of microtubules at molar ratios that could be achieved locally within the cell. Our results are consistent with the hypothesis that calmodulin may be controlling microtubule polymerization equilibria in areas of high local concentration such as the mitotic spindle.A large variety of cellular events including mitosis, cell elongation, and neurite outgrowth are believed to be related to the polymerization and depolymerization of microtubules in vivo (1) . However, the mechanisms by which these events might be regulated are only begining to be understood, with the bulk of the evidence drawn from in vitro biochemical studies .A number ofinvestigators (2) have found that microtubules in crude extracts are depolymerized by concentrations of calcium much lower than those required to depolymerize tubules made from purified microtubule proteins. This disparity suggested that some factor in crude extracts "sensitized" the tubules to calcium ; calmodulin was an obvious candidate for this function . When calmodulin was added to purified microtubule proteins, Marcum et al. (3) found that the calcium concentration required for microtubule depolymerization was decreased by two orders of magnitude, to approximately the level observed in crude extracts. In addition it was found that the inhibition of microtubule polymerization was proportional both to calmodulin and to calcium concentrations (3) . While this interaction occurs at calmodulin:tubulin in ratios much higher than the ratios of calmodulin :enzyme necessary to activate cellular enzymes, at least two factors argue in favor of its physiological relevance. First, the im- 1918CHARLES KEITH, MARIO DiPAOLA, FREDERICK R. MAXFIELD, and MICHAEL L. SHELANSKI Department of Pharmacology, New York University Medical Center, New York 10016 munocytochemical localization of calmodulin between the chromosomes and spindle poles of mitotic cells (4, 5) and at the postsynaptic termini of basal ganglion neurons (6) correlates with the locations in the cell where microtubule depolymerization might be expected to occur...

Microtubule proteins and tubulin have been purified from brain and labeled with dichlorotriazinyl fluorescein (DTAF). This procedure compromises neither the polymerizability of the proteins nor their affinities for unlabeled proteins. Within 15 min after microinjection of either DTAF-microtubule proteins or DTAF-tubulin into cultured gerbil fibroma cells, there was an evolution of a fluorescent fibrillar pattern with a distribution similar to that of the microtubular network seen after staining with fluorescent antitubulin. These filaments were colchicine sensitive and could be seen to elongate with time. DTAF-labeled microtubule accessory proteins from brain were not incorporated into filaments and appeared to label autophagic vacuoles.

Cytoplasmic free calcium has been proposed as a regulator of many microtubule-mediated processes, including mitosis. It has been difficult to test this hypothesis because methods for local measurement of free Ca2+ in the living cell have not been available. We have used the fluorescent calcium indicator dye Quin-2 (methoxyquinoline-1bis(o-aminophenoxy)ethane-N,N,N',N' -tetra acetic acid), which allows such observations to be made by digital processing of fluorescent images from the light microscope. Here we report the application of this technique to the study of local Ca2+ concentrations in mitotic endosperm cells of Haemanthus sp., and show that there is transient increase in free Ca2+ at the mitotic spindle poles during anaphase. This locally high Ca2+ may provide a mechanism for the regional control of microtubules and other cytoskeletal elements during anaphase.