Characterization of the first FGFRL1 mutation identified in a craniosynostosis patient

Fibroblast growth factor receptor-like 1 (FGFRL1) is a recently discovered transmembrane protein whose functions remain unclear. Since mutations in the related receptors FGFR1-3 cause skeletal malformations, DNA samples from 55 patients suffering from congenital skeletal malformations and 109 controls were searched for mutations in FGFRL1. One patient was identified harboring a frameshift mutation in the intracellular domain of this novel receptor. The patient showed craniosynostosis, radio-ulnar synostosis and genital abnormalities and had previously been diagnosed with Antley-Bixler syndrome. The effect of the FGFRL1 mutation was studied in vitro. In a reporter gene assay, the wild-type as well as the mutant receptor inhibited FGF signaling. However, the mutant protein differed from the wild-type protein in its subcellular localization. Mutant FGFRL1 was mainly found at the plasma membrane where it interacted with FGF ligands, while the wild-type protein was preferentially located in vesicular structures and the Golgi complex. Two motifs from the intracellular domain of FGFRL1 appeared to be responsible for this differential distribution, a tandem tyrosine based motif and a histidine-rich sequence. Deletion of either one led to the preferential redistribution of FGFRL1 to the plasma membrane. It is therefore likely that mutant FGFRL1 contributes to the skeletal malformations of the patient.

Characterization of three human sec14p-like proteins: alpha-tocopherol transport activity and expression pattern in tissues

Three closely related human sec14p-like proteins (hTAP1, 2, and 3, or SEC14L2, 3, and 4, respectively) have been described. These proteins may participate in intracellular lipid transport (phospholipids, squalene, tocopherol analogues and derivatives) or influence regulatory lipid-dependent events. Here, we show that the three recombinant hTAP proteins associate with the Golgi apparatus and mitochondria, and enhance the in vitro transport of radioactively labeled alpha-tocopherol to mitochondria in the same order of magnitude as the human alpha-tocopherol transfer protein (alpha-TTP). hTAP1 and hTAP2 are expressed in several cell lines, whereas the expression level of hTAP3 is low. Expression of hTAP1 is induced in human umbilical cord blood-derived mast cells upon differentiation by interleukin 4. In tissues, the three hTAPs are detectable ubiquitously at low level; pronounced and localized expression is found for hTAP2 and hTAP3 in the perinuclear region in cerebellum, lung, liver and adrenal gland. hTAP3 is well expressed in the epithelial duct cells of several glands, in ovary in endothelial cells of small arteries as well as in granulosa and thecal cells, and in testis in Leydig cells. Thus, the three hTAPs may mediate lipid uptake, secretion, presentation, and sub-cellular localization in a tissue-specific manner, possibly using organelle- and enzyme-specific docking sites.

Chondrocyte mechanotransduction: effects of compression on deformation of intracellular organelles and relevance to cellular biosynthesis

OBJECTIVE: The effects of mechanical deformation of intact cartilage tissue on chondrocyte biosynthesis in situ have been well documented, but the mechanotransduction pathways that regulate such phenomena have not been elucidated completely. The goal of this study was to examine the effects of tissue deformation on the morphology of a range of intracellular organelles which play a major role in cell biosynthesis and metabolism. DESIGN: Using chemical fixation, high pressure freezing, and electron microscopy, we imaged chondrocytes within mechanically compressed cartilage explants at high magnification and quantitatively and qualitatively assessed changes in organelle volume and shape caused by graded levels of loading. RESULTS: Compression of the tissue caused a concomitant reduction in the volume of the extracellular matrix (ECM), chondrocyte, nucleus, rough endoplasmic reticulum, and mitochondria. Interestingly, however, the Golgi apparatus was able to resist loss of intraorganelle water and retain a portion of its volume relative to the remainder of the cell. These combined results suggest that a balance between intracellular mechanical and osmotic gradients govern the changes in shape and volume of the organelles as the tissue is compressed. CONCLUSIONS: Our results lead to the interpretive hypothesis that organelle volume changes appear to be driven mainly by osmotic interactions while shape changes are mediated by structural factors, such as cytoskeletal interactions that may be linked to extracellular matrix deformations. The observed volume and shape changes of the chondrocyte organelles and the differential behavior between organelles during tissue compression provide evidence for an important mechanotransduction pathway linking translational and post-translational events (e.g., elongation and sulfation of glycosaminoglycans (GAGs) in the Golgi) to cell deformation.

Compound heterozygous mutations affect protein folding and function in patients with congenital sucrase-isomaltase deficiency

BACKGROUND & AIMS: Congenital sucrase-isomaltase (SI) deficiency is an autosomal-recessive intestinal disorder characterized by a drastic reduction or absence of sucrase and isomaltase activities. Previous studies have indicated that single mutations underlie individual phenotypes of the disease. We investigated whether compound heterozygous mutations, observed in some patients, have a role in disease pathogenesis. METHODS: We introduced mutations into the SI complementary DNA that resulted in the amino acid substitutions V577G and G1073D (heterozygous mutations found in one group of patients) or C1229Y and F1745C (heterozygous mutations found in another group). The mutant genes were expressed transiently, alone or in combination, in COS cells and the effects were assessed at the protein, structural, and subcellular levels. RESULTS: The mutants SI-V577G, SI-G1073D, and SI-F1745C were misfolded and could not exit the endoplasmic reticulum, whereas SI-C1229Y was transported only to the Golgi apparatus. Co-expression of mutants found on each SI allele in patients did not alter the protein's biosynthetic features or improve its enzymatic activity. Importantly, the mutations C1229Y and F1745C, which lie in the sucrase domains of SI, prevented its targeting to the cell's apical membrane but did not affect protein folding or isomaltase activity. CONCLUSIONS: Compound heterozygosity is a novel pathogenic mechanism of congenital SI deficiency. The effects of mutations in the sucrase domain of SIC1229Y and SIF1745C indicate the importance of a direct interaction between isomaltase and sucrose and the role of sucrose as an intermolecular chaperone in the intracellular transport of SI.

Intracellular localization of the BCL-2 family member BOK and functional implications

The pro-apoptotic BCL-2 family member BOK is widely expressed and resembles the multi-BH domain proteins BAX and BAK based on its amino acid sequence. The genomic region encoding BOK was reported to be frequently deleted in human cancer and it has therefore been hypothesized that BOK functions as a tumor suppressor. However, little is known about the molecular functions of BOK. We show that enforced expression of BOK activates the intrinsic (mitochondrial) apoptotic pathway in BAX/BAK-proficient cells but fails to kill cells lacking both BAX and BAK or sensitize them to cytotoxic insults. Interestingly, major portions of endogenous BOK are localized to and partially inserted into the membranes of the Golgi apparatus as well as the endoplasmic reticulum (ER) and associated membranes. The C-terminal transmembrane domain of BOK thereby constitutes a 'tail-anchor' specific for targeting to the Golgi and ER. Overexpression of full-length BOK causes early fragmentation of ER and Golgi compartments. A role for BOK on the Golgi apparatus and the ER is supported by an abnormal response of Bok-deficient cells to the Golgi/ER stressor brefeldin A. Based on these results, we propose that major functions of BOK are exerted at the Golgi and ER membranes and that BOK induces apoptosis in a manner dependent on BAX and BAK.

The role of zinc dynamics in growth hormone secretion

Human growth hormone (GH) causes a variety of physiological and metabolic effects in humans and plays a pivotal role in postnatal growth. In somatotroph cells of the anterior pituitary, GH is stored in concentrated forms in secretory granules to be rapidly released upon GH-releasing hormone stimulation. During the process of secretory granule biogenesis, self-association of GH occurs in the compartments of the early secretory pathway (endoplasmic reticulum and Golgi complex). Since this process is greatly facilitated by the presence of zinc ions, it is of importance to understand the potential role of zinc transporters that participate in the fine-tuning of zinc homeostasis and dynamics, particularly in the early secretory pathway. Thus, the role of zinc transporters in supplying the secretory pathway with the sufficient amount of zinc required for the biogenesis of GH-containing secretory granules is essential for normal secretion. This report, illustrated by a clinical case report on transient neonatal zinc deficiency, focuses on the role of zinc in GH storage in the secretory granules and highlights the role of specific zinc transporters in the early secretory pathway.

Post-translational regulation of an Aplysia glutamate transporter during long-term facilitation.

Regulation of glutamate transporters accompanies plasticity of some glutamatergic synapses. The regulation of glutamate uptake at the Aplysia sensorimotor synapse during long-term facilitation (LTF) was investigated. Previously, increases in levels of ApGT1 (Aplysia glutamate transporter 1) in synaptic membranes were found to be related to long-term increases in glutamate uptake. In this study, we found that regulation of ApGT1 during LTF appears to occur post-translationally. Serotonin (5-HT) a transmitter that induces LTF did not increase synthesis of ApGT1. A pool of ApGT1 appears to exist in sensory neuron somata, which is transported to the terminals by axonal transport. Blocking the rough endoplasmic reticulum-Golgi-trans-Golgi network (TGN) pathway with Brefeldin A prevented the 5-HT-induced increase of ApGT1 in terminals. Also, 5-HT produced changes in post-translational modifications of ApGT1 as well as changes in the levels of an ApGT1-co-precipitating protein. These results suggest that regulation of trafficking of ApGT1 from the vesicular trafficking system (rough endoplasmic reticulum-Golgi-TGN) in the sensory neuron somata to the terminals by post-translational modifications and protein interactions appears to be the mechanism underlying the increase in ApGT1, and thus, glutamate uptake during memory formation.

Long-term regulation of neuronal high-affinity glutamate and glutamine uptake in Aplysia.

An increase in transmitter release accompanying long-term sensitization and facilitation occurs at the glutamatergic sensorimotor synapse of Aplysia. We report that a long-term increase in neuronal Glu uptake also accompanies long-term sensitization. Synaptosomes from pleural-pedal ganglia exhibited sodium-dependent, high-affinity Glu transport. Different treatments that induce long-term enhancement of the siphon-withdrawal reflex, or long-term synaptic facilitation increased Glu uptake. Moreover, 5-hydroxytryptamine, a treatment that induces long-term facilitation, also produced a long-term increase in Glu uptake in cultures of sensory neurons. The mechanism for the increase in uptake is an increase in the V(max) of transport. The long-term increase in Glu uptake appeared to be dependent on mRNA and protein synthesis, and transport through the Golgi, because 5,6-dichlorobenzimidazole riboside, emetine, and brefeldin A inhibited the increase in Glu uptake. Also, injection of emetine and 5,6-dichlorobenzimidazole into Aplysia prevented long-term sensitization. Synthesis of Glu itself may be regulated during long-term sensitization because the same treatments that produced an increase in Glu uptake also produced a parallel increase in Gln uptake. These results suggest that coordinated regulation of a number of different processes may be required to establish or maintain long-term synaptic facilitation.

Studies on the {\it in vivo} function of a phosphatidylinositol transfer protein from {\it Saccharomyces cerevisiae}

Phosphatidylinositol transfer proteins (PI-TP's) catalyze the transfer of phosphatidylinositol and phosphatidylcholine between membranes in vitro. However the in vivo function of these proteins is unknown. In this thesis we have used a combined biochemical and genetic approach to determine the importance of PI-TP in vivo. An oligonucleotide based on the amino terminal sequence of the PI-TP from Saccharomyces cerevisiae, was used to screen a yeast genomic library for the gene encoding PI-TP (PIT1 gene). Yeast strains transformed with the positive clones showed overproduction of transfer activities and transfer protein in the 100,000 x g supernatants. The 5$\sp\prime$ terminus of the PIT1 gene correlates with the predicted codons for residues 3-30 of the determined protein sequence. Tetrad analysis of a heterozygous diploid (PIT1/pit1::LEU2) revealed that the PIT1 gene is essential for cell growth. Non-viable spores could be rescued by transformation of the above diploid prior to sporulation, with a plasmid borne copy of the wild type gene. Sequencing of the entire PIT1 gene has revealed that the PIT1 gene is identical to the SEC14 gene. The sec14 ts mutant which exhibits conditional defects at the Golgi stage of protein secretion, is also temperature sensitive for PI-TP activity in vitro. These findings represent the first instance in which a physiological function has been assigned to any phospholipid transfer protein. ^

Function and formation of $\beta$1,4-galactosyltransferase-specific adhesions during growth and differentiation of F9 embryonal carcinoma cells

Galactosyltransferase (GalTase) is localized in the Golgi, where it functions in oligosaccharide synthesis, as well as on the cell surface where it serves as a cell adhesion molecule. GalTase-specific adhesions are functional in a number of important biological events, including F9 embryonal carcinoma (EC) cell adhesions. GalTase-based adhesions are formed by recognition and binding to terminal N-acetylglucosamine (GlcNAc) residues on its glycoprotein counterpart on adjacent cell surfaces. The object of this work has been to investigate the formation and function of GalTase-specific adhesions during F9 cell growth and differentiation. We initially investigated GalTase synthesis during differentiation and found that the increase in GalTase activity was specific for the Golgi compartment; surface GalTase levels remained constant during differentiation. These data indicated that the increase in cell adhesions expected with increased cell-matrix interaction in differentiated F9 cells is not the consequence of increased surface GalTase expression and, more interestingly, that the two pools of GalTase are under differential regulation. Synthesis and recognition of the consociate glycoprotein component was next investigated. Surface GalTase recognized several surface glycoproteins in a pattern that changes with differentiation. Uvomorulin, lysosome-associated membrane protein-1 (LAMP-1), and laminin were recognized by surface GalTase and are, therefore, potential components in GalTase-specific adhesions. Furthermore, these interactions were aberrant in an adhesion-defective F9 cell line that results, at least in part, from abnormal oligosaccharide synthesis. The function played by surface GalTase in growth and induction of differentiation was examined. Inhibition of surface GalTase function by a panel of reagents inhibited F9 cell growth. GalTase expression at both the transcription and protein levels were differentially regulated during the cell cycle, with surface expression greatest in the G1 phase. Disruption of GalTase adhesion by exposure to anti-GalTase antibodies during this period resulted in extension of the G2 phase, a result similar to that seen with agents known to inhibit growth and induce differentiation. Finally, other studies have suggested that a subset of cell adhesion molecules have the capability to induce differentiation in EC cells systems. We have determined in F9 cells that dissociating GalTase adhesion by galactosylation of and release of the consociate glycoproteins induces differentiation, as defined by increased laminin synthesis. The ability to induce differentiation by surface galactosylation was greatest in cells grown in cultures promoting cell-cell adhesions, relative to cultures with minimal cell-cell interactions. ^

Molecular analysis of $\beta$1,4-galactosyl-transferase localization to the cell surface and participation in cell adhesion

$\beta$1,4-Galactosyltransferase (GalTase) is unusual among the glycosyltransferases in that it is found in two subcellular compartments where it performs different functions. In the trans-Golgi complex, GalTase participates in oligosaccharide biosynthesis as do other glycosyltransferases. GalTase is also found on the cell surface, where it associates with the cytoskeleton and functions as a receptor for extracellular oligosaccharide ligands. Although we know much regarding GalTase function on the cell surface, little is known about the mechanisms underlying its transport to the plasma membrane. Cloning of the GalTase gene revealed that there are two GalTase proteins (i.e., long and short) with different size cytoplasmic tails. This raises the possibility that differences in the cytoplasmic domain of GalTase may influence its subcellular distribution. The object of this study was to examine this hypothesis directly through the use of molecular, immunological, and biochemical approaches.^ To examine whether the two GalTase proteins are targeted to different subcellular compartments, F9 embryonal carcinoma cells were transfected with either long or short GalTase cDNAs and intracellular and cell surface enzyme levels measured. Cell surface GalTase activity was enriched in cells overexpressing the long, but not the form of short GalTase. Furthermore, a dominant negative mutation in cell surface GalTase was created by transfecting cells with GalTase cDNAs encoding a truncated version of long GalTase devoid of the extracellular catalytic domain. Overexpressing the complete cytoplasmic and transmembrane domains of long GalTase led to a loss of GalTase-dependent cellular adhesion by specifically displacing surface GalTase from its cytoskeletal associations. In contrast, overexpressing the analogous truncated protein of short GalTase had no effect on cell adhesion. Finally, chloramphenicol acetyltransferase (CAT) reporter proteins were used to determine directly whether the cytoplasmic domains of long and short GalTase were responsible for differential subcellular distribution. The cytoplasmic and transmembrane domains of long GalTase led to CAT expression on the ceil surface and its association with the detergent-insoluble cytoskeleton; the analogous fusion protein containing short GalTase was restricted to the Golgi compartment. These results suggest that the cytoplasmic domain unique to long GalTase is responsible for targeting a portion of this protein to the cell surface and associating it with the cytoskeleton, enabling it to function as a cell adhesion molecule. ^

Defective pituitary function and gamete interactions in $\beta$1,4-galactosyltransferase null mice

Despite much attention, the function of oligosaccharide chains of glycoproteins remains largely unknown. Our understanding of oligosaccharide function in vivo has been limited to the use of reagents and targeted mutations that eliminate entire oligosaccharide chains. However, most, if not all biological functions for oligosaccharides have been attributed to specific terminal sequences on these oligosaccharides, yet there have been few studies to examine the consequences of modifying terminal oligosaccharide structures in vivo. To address this issue, mice were created bearing a targeted mutation in $\beta$1,4-galactosyltransferase, an enzyme responsible for elaboration of many of the proposed biologically-active carbohydrate epitopes. Most galactosyltransferase-null mice died within the first few weeks after birth and were characterized by stunted growth, thin skin, sparse hair, and dehydration. In addition, the adrenal cortices were poorly stratified and spermatogenesis was delayed. The few surviving adults had puffy skin (myxedema), difficulty delivering pups at birth (dystocia), and failed to lactate (agalactosis). All of these defects are consistant with endocrine insufficiency, which was confirmed by markedly decreased levels of serum thyroxine. The anterior pituitary gland appeared functionally delayed in newborn mutant mice, since the constituent cells were quiescent and nonsecretory, unlike that of control littermates. However, the anterior pituitary acquired a normal secretory phenotype during neonatal development, although it remained abnormally small and its glycoprotein hormones were devoid of $\beta$1,4-galactosyl residues. These results support in vitro studies suggesting that incomplete glycosylation of pituitary hormones leads to the creation of hormone antagonists that down regulate subsequent endocrine function producing polyglandular endocrine insufficiency. More surprisingly, the fact that some mice survive this neonatal period indicates the presence of a previously unrecognized compensatory pathway for glycoprotein hormone glycosylation and/or action.^ In addition to its well-studied biosynthetic function in the Golgi complex, a GalTase isoform is also expressed on the sperm surface where it functions as a gamete receptor during fertilization by binding to its oligosaccharide ligand on the egg coat glycoprotein, ZP3. Aggregation of GalTase by multivalent ZP3 oligosaccharides activates a G-protein cascade leading to the acrosome reaction. Although GalTase-null males are fertile, the mutant sperm bind less ZP3 than wild-type sperm, and are unable to undergo the acrosome reaction in response to either zona pellucida glycoproteins or to anti-GalTase anti-serum, as do wild-type sperm. However, mutant and wild-type sperm undergo the acrosome reaction normally in response to calcium ionophore which bypasses the requirement for ZP3 binding. Interestingly, the phenotype of the GalTase-null sperm is reciprocal to that of sperm that overexpress surface GalTAse and which bind more ZP3 leading to precocious acrosome reactions. These results confirm that GalTase functions as at least one of the sperm receptors for ZP3, and that GalTase participates in the ZP3-induced signal transduction pathway during zona pellucida-induced acrosome reactions. ^

IMMUNOLOGICAL AND CYTOLOGICAL INVESTIGATION OF GAMMA INTERFERON PRODUCTION IN HUMAN T-LYMPHOCYTES INDUCED BY PMA AND PHA (IMMUNOGOLD, CELL CYCLE ANALYSIS)

The present study examined cellular mechanisms involved in the production and secretion of human (gamma)IFN. The hypothesis of this investigation was that (gamma)IFN is an export glycoprotein whose synthesis in human T lymphocytes is dependent on membrane stimulation, polypeptide synthesis in the rough endoplasmic reticulum, packaging in the Golgi complex, and release from the cell by exocytosis.^ The model system for this examination utilized T lymphocytes from normal donors and patients with chronic lymphocytic leukemia (CLL) induced in vitro with the tumor promoter, phorbol 12-myristate 13-acetate (PMA) and the lectin, phytohemagglutinin (PHA) to produce (gamma)IFN. This study reconfirmed the ability of PMA and PHA to synergistically induce (gamma)IFN production in normal T lymphocytes, as measured by viral inhibition assays and radio-immunoassays for (gamma)IFN. The leukemic T cells were demonstrated to produce (gamma)IFN in response to treatment with PHA. PMA treatment also induced (gamma)IFN production in the leukemic T cells, which was much greater than that observed in similarly treated normal T cells. In these same cells, however, combined treatment of the agents was shown to be ineffective at inducing (gamma)IFN production beyond the levels stimulated by the individual agents. In addition, the present study reiterated the synergistic effect of PMA/PHA on the stimulation of growth kinetics in normal T cells. The cell cycle of the leukemic T cells was also responsive to treatment with the agents, particularly with PMA treatment. A number of morphological alterations were attributed to PMA treatment including the acquisition of an elongated configuration, nuclear folds, and large cytoplasmic vacuoles. Many of the effects were observed to be reversible with dilution of the agents, and reversion to this state occurred more rapidly in the leukemic T cells. Most importantly, utilization of a thin section immuno-colloidal gold labelling technique for electron microscopy provided, for the first time, direct evidence of the cellular mechanism of (gamma)IFN production and secretion. The results of this latter study support the idea that (gamma)IFN is produced in the rough endoplasmic reticulum, transferred to the Golgi complex for accumulation and packaging, and released from the T cells by exocytosis. ^

Clathrin heavy chain plays multiple roles in polarizing the Drosophila oocyte downstream of Bic-D

Bicaudal-D (Bic-D), Egalitarian (Egl), microtubules and their motors form a transport machinery that localizes a remarkable diversity of mRNAs to specific cellular regions during oogenesis and embryogenesis. Bic-D family proteins also promote dynein-dependent transport of Golgi vesicles, lipid droplets, synaptic vesicles and nuclei. However, the transport of these different cargoes is still poorly understood. We searched for novel proteins that either mediate Bic-Ddependent transport processes or are transported by them. Clathrin heavy chain (Chc) co-immunopurifies with Bic-D in embryos and ovaries, and a fraction of Chc colocalizes with Bic-D. Both proteins control posterior patterning of the Drosophila oocyte and endocytosis. Although the role of Chc in endocytosis is well established, our results show that Bic-D is also needed for the elevated endocytic activity at the posterior of the oocyte. Apart fromaffecting endocytosis indirectly by its role in osk mRNA localization, Bic-D is also required to transport Chc mRNA into the oocyte and for transport and proper localization of Chc protein to the oocyte cortex, pointing to an additional,more direct role of Bic-D in the endocytic pathway. Furthermore, similar to Bic-D, Chc also contributes to proper localization of osk mRNA and to oocyte growth. However, in contrast to other endocytic components and factors of the endocytic recycling pathway, such as Rabenosyn-5 (Rbsn-5) and Rab11, Chc is needed during early stages of oogenesis (from stage 6 onwards) to localize oskmRNA correctly.Moreover,we also uncovered a novel, presumably endocytosis-independent, role of Chc in the establishment of microtubule polarity in stage 6 oocytes.

Fine structure of synapses on dendritic spines

Camillo Golgi's "Reazione Nera" led to the discovery of dendritic spines, small appendages originating from dendritic shafts. With the advent of electron microscopy (EM) they were identified as sites of synaptic contact. Later it was found that changes in synaptic strength were associated with changes in the shape of dendritic spines. While live-cell imaging was advantageous in monitoring the time course of such changes in spine structure, EM is still the best method for the simultaneous visualization of all cellular components, including actual synaptic contacts, at high resolution. Immunogold labeling for EM reveals the precise localization of molecules in relation to synaptic structures. Previous EM studies of spines and synapses were performed in tissue subjected to aldehyde fixation and dehydration in ethanol, which is associated with protein denaturation and tissue shrinkage. It has remained an issue to what extent fine structural details are preserved when subjecting the tissue to these procedures. In the present review, we report recent studies on the fine structure of spines and synapses using high-pressure freezing (HPF), which avoids protein denaturation by aldehydes and results in an excellent preservation of ultrastructural detail. In these studies, HPF was used to monitor subtle fine-structural changes in spine shape associated with chemically induced long-term potentiation (cLTP) at identified hippocampal mossy fiber synapses. Changes in spine shape result from reorganization of the actin cytoskeleton. We report that cLTP was associated with decreased immunogold labeling for phosphorylated cofilin (p-cofilin), an actin-depolymerizing protein. Phosphorylation of cofilin renders it unable to depolymerize F-actin, which stabilizes the actin cytoskeleton. Decreased levels of p-cofilin, in turn, suggest increased actin turnover, possibly underlying the changes in spine shape associated with cLTP. The findings reviewed here establish HPF as an appropriate method for studying the fine structure and molecular composition of synapses on dendritic spines.