The nature of second messenger-responsive intracellular Ca2+ stores in neurons remains open for discussion. Here, we demonstrate the existence in Purkinje cells (PCs) of endoplastic reticulum (ER) subcompartments characterized by an uneven distribution of three proteins involved in Ca2+ storage and release: the inositol 1,4,5-trisphosphate (InsP3) receptor, Ca(2+)-ATPase, and calsequestrin. Ca(2+)-ATPase and the InsP3 receptor have a widespread, although not identical, distribution throughout the ER. Calsequestrin is localized throughout the smooth ER and is particularly concentrated in pleiomorphic vesicles with a moderately electron-dense core, which appear to represent a subcompartment of the smooth ER. In double-labeling experiments many of these vesicles were unlabeled by InsP3 receptor antibodies. These results suggest a key role of the ER as an intracellular Ca2+ store and demonstrate a possible structural basis for distinct intracellular Ca2+ pools regulated by different second messengers.

Abstract. The pathways of synaptic vesicle (SV) biogenesis and recycling are still poorly understood. We have studied the effects of Brefeldin A (BFA) on the distribution of several SV membrane proteins (synaptophysin, synaptotagmin, synaptobrevin, p29, SV2 and rab3A) and on endosomal markers to investigate the relationship between SVs and the membranes with which they interact in cultured hippocampal neurons developing in isolation. In these neurons, SV proteins are detected as punctate immunoreactivity that is concentrated in axons but is also present in perikarya and dendrites. In the same neurons, the transferrin receptor, a well established marker of early endosomes, is selectively concentrated in perikarya and dendrites. In the perikaryal-dendritic region, BFA induced a dramatic tubulation of transferrin receptors as well as a cotubulation of the bulk of synaptophysin. Synaptotagrain, synaptobrevin, p29 and SV2 immunoreactivities retained a primarily punctate distribution. No tubulation of rab3A was observed. In axons, BFA did not produce any obvious alteration of the distribution of SV proteins, nor of peroxidase-or Lucifer yellowlabeled early endosomes. The selective effect of BFA on dendritic membranes suggests the existence of functional differences between the endocytic systems in dendrites and axons. Cotubulation of transferrin receptors and synaptophysin in the perikaryal-dendritic region is consistent with a functional interconnection between the traffic of SV proteins and early endosomes. The heterogeneous effects of BFA on SV proteins in this cell region indicates that SV proteins are differentially sorted upon exit from the TGN and are coassembled into SVs at the cell periphery.

Mutations in the hook gene alter intracellular trafficking of internalized ligands in Drosophila. To dissect this defect in more detail, we developed a new approach to visualize the pathway taken by the Bride of Sevenless (Boss) ligand after its internalization into R7 cells. A chimeric protein consisting of HRP fused to Boss (HRP-Boss) was expressed in R8 cells. This chimera was fully functional: it rescued the boss mutant phenotype, and its trafficking was indistinguishable from that of the wild-type Boss protein. The HRP activity of the chimera was used to follow HRP-Boss trafficking on the ultrastructural level through early and late endosomes in R7 cells. In both wild-type and hook mutant eye disks, HRP-Boss was internalized into R7 cells. In wild-type tissue, Boss accumulated in mature multivesicular bodies (MVBs) within R7 cells; such accumulation was not observed in hook eye disks, however. Quantitative electron microscopy revealed a loss of mature MVBs in hook mutant tissue compared with wild type, whereas more than twice as many multilammelar late endosomes were detected. Our genetic analysis indicates that Hook is required late in endocytic trafficking to negatively regulate delivery from mature MVBs to multilammelar late endosomes and lysosomes. INTRODUCTIONEukaryotic cells carefully regulate trafficking of internalized proteins (reviewed in Mukherjee et al., 1997). In most cells, endocytic vesicles deliver internalized cargo to early endosomes from which many proteins are shuttled back to the plasma membrane through a recycling compartment (Ghosh et al., 1994). Other proteins accumulate in vacuolar subcompartments of early endosomes before their transformation into mature multivesicular bodies (MVBs) 1 named for their characteristic morphology (Stoorvogel et al., 1991;Dunn and Maxfield, 1992;van Deurs et al., 1993;Futter et al., 1996). From MVBs, endocytic cargo is delivered to morphologically distinct prelysosomal structures, the multilammelar late endosomes (Gruenberg and Maxfield, 1995;van Deurs et al., 1995;Futter et al., 1996;Mullock et al., 1998). The biochemical mechanisms that regulate these trafficking events late in the endocytic pathway are not well understood (Mukherjee et al., 1997).The genetic dissection of endocytosis and vacuolar delivery in yeast has contributed much to our understanding of the underlying biochemical mechanisms (Wendland et al., 1998). Many genes have been identified that are required in the early and late phases of trafficking from the yeast cell surface to the vacuole (Riezman, 1993;Stack et al., 1995). Different subclasses of these genes affect specific steps along the endocytic and biosynthetic pathways, and their conservation suggests similar roles for their mammalian counterparts (Horazdovsky et al., 1994;Odorizzi et al., 1998;Sato et al., 1998 Mutations in Drosophila constitute a resource for the genetic dissection of endocytic trafficking in multicellular organisms (Lloyd et al., 1998). Notably, the discovery that the shibire gene encodes the Drosophila homo...

The internal ear's sensory receptor, or hair cell, responds when stimuli deflect its mechanoreceptive hair bundle. As a hair cell adapts to sustained stimulation, mechanical adjustments within the bundle reset its position of sensitivity. Because several lines of experimentation suggest that a form of myosin I mediates adaptation, we endeavored to clone cDNAs ending this motor molecule. By using degenerate oligonucleotide primers based upon the deduced amino acid sequence for mammalian myosin 13, we performed reverse transcription and polymerase chain reactions (PCRs) to produce a candidate cDNA from polyadenylylated mRNA isolated from the frog's brain. The resultant product was used to probe a cDNA library, from which were isolated clones encoding an =-119-kDa isozyme of myosin Id. PCR amplification disclosed the presence of mRNA encoding the same isozyme in tissue from the bullfrog's sacculus, an organ of the internal ear. When expressed as a bacterial fusion protein, a domain from the tail region of this form of myosin I was recognized by monoclonal antibodies that react with myosin I in hair bundles.This cloned l19-kDa isozyme of myosin I is accordingly a candidate to be the motor molecule responsible for the adaptation of mechanoelectrical transduction by hair cells.