Developmental Changes in the Structure and Composition of the Postsynaptic Density

The development of the brain and its underlying circuitry is dependent on the formation of trillions of chemical synapses, which are highly specialized contacts that regulate the flow of information from one neuron to the next. It is through these synaptic connections that neurons wire together into networks capable of performing specific tasks, and activity-dependent changes in their structural and physiological state is one way that the brain is thought to adapt and store information. At the ultrastructural level, developmental and activity-dependent changes in the size and shape of dendritic spines have been well documented, and it is widely believed that structural changes in spines are a hallmark sign of synapse maturation and alteration of synaptic physiology. While changes in spine structure have been studied extensively, changes in one of its most prominent components, the postsynaptic density (PSD), have largely evaded observation. The PSD is a protein-rich organelle on the cytoplasmic side of the postsynaptic membrane, where it sits in direct opposition to the presynaptic terminal. The PSD functions both to cluster neurotransmitter receptors at the cell surface as well as organize the intracellular signaling molecules responsible for transducing extracellular signals to the postsynaptic cell. Much is known about the chemical composition of the PSD, but the structural arrangement of its molecular components is not well documented. Adding to the difficulty of understanding such a complex mass of protein machinery is the fact that its protein composition is known to change in response to synaptic activity, meaning that its structure is plastic and no two PSDs are identical. Here, immuno-gold labeling and electron tomography of PSDs isolated throughout development was used to track changes in both the structure and molecular composition of the PSD. State-of-the-art cryo-electron tomography was used to study the fine structure of the PSD during development, and provides an unprecedented glimpse into its molecular architecture in an un-fixed, unstained and hydrated state. Through this analysis, large structural and compositional changes are apparent and suggest a model by which the PSD is first assembled as a mesh-like lattice of proteins that function as support for the later recruitment of various PSD components. Spatial analysis of the recruitment of proteins into the PSD demonstrated that its assembly has an underlying order.

Motile response patterns and ultrastructural observations of the isolated outer hair cell

The tonotopic organization of the mammalian cochlea is accompanied by structural gradients which include the somatic lengths of outer hair cells (OHCs). These receptors rest upon the vibrating portion of the basilar membrane and have been reported to exhibit motile responses following chemical and electrical stimulation. These movements were examined in detail in this dissertation. It was found that isolated OHCs cultured in vitro respond to chemical depolarization with slow tonic movements, and to electrical waveforms with bi-directional, frequency following movements extending from DC to at least 10 kHz.^ Slow contractions were also elicited following electrical stimulation, bath incubation in carbachol (a cholinergic agonist), and increases in extracellular K+ concentration as little as 50 mM.^ Isolated OHCs display anatomical features which are remarkable when contrasted with those prepared from intact receptor organs. A complex structure located between the cuticular plate and the nuclear membrane was consistently observed and was examined by serial cross-sections which revealed a network of non-membrane bound densities. This corresponded to a granular complex seen at the light microscope level. The complex was composed of dense regions of organelles, striated structures embedded within the core, and a circumferential network of microtubules residing in the peri-nuclear portion of the cell. In cells which had lost their nuclear attachment to the terminal synaptic body, the granular complex could be made to contract without effecting any change in cellular length, implying that the complex may be the driving force behind certain aspects of the motile response.^ Most cells displayed movements which revealed asymmetries analogous to those reported for OHC receptor potentials in vivo. The contraction phase (for longer cells) was shown to have a small time constant (approximately 400 microseconds) and saturated with limited displacements. The expansion phase had time constants as large as 1.3 milliseconds but yielded displacements as much as 60 percent larger than those seen for contractions.^ Additional waveform characteristics seen in the in vivo response could be emulated either by biasing the cell's resting length with either direct current, triggering contractions via large electrical displacements, or incubation with depolarizing compounds.^ Alternatively, short (20-30 um) cells revealed more linear response characteristics to the probe stimulus. Partial saturation was achieved and revealed a DC component which was opposite in polarity to that seen in longer cells. (Abstract shortened with permission of author.) ^

CYTOLOGICAL, ULTRASTRUCTURAL, AND BIOCHEMICAL STUDIES OF THE STRUCTURE OF PREMATURELY CONDENSED CHROMOSOMES

The phenomenon of premature chromosome condensation, resulting from fusion between mitotic and interphase cells, includes dissolution of the interphase nuclear framework, thus allowing a direct visualization of interphase chromosomes. Light microscope morphology of prematurely condensed chromosomes (PCC) from synchronized HeLa cells supports the model of an interphase "chromosome condensation cycle". PCC are increasingly attenuated as cells progress through G(,1). A maximum degree of decondensation is observed at active sites of DNA replication during S phase, and a condensed morphology is rapidly resumed following completion of replication of a chromosome segment.^ To permit ultrastructural and biochemical studies of PCC, a procedure was developed to induce premature chromosome condensation at high frequency. This was achieved by polyethylene glycol (PEG)-mediated fusion of a dense monolayer of mitotic and interphase cells induced by centrifugation onto lectin-coated culture dishes. Using this method, PCC induction frequencies of 60-90% are routinely obtained.^ Scanning electron microscope analysis of PCC spreads revealed that the extension of PCC during progression through G(,1) is accompanied by a transition of the basic 30 nm chromatin fiber from tightly packed looping fibers to extended longitudinal fibers. Sites of active DNA replication is S-PCC were indicated to be organized a single longitudinal fibers. Following replication of a chromosome segment, a rapid reorganization from the extended longitudinal fiber to packed looping fibers occurs. The postreplication maturation process appears to include the assembly of a chromosome core consisting of multiple longitudinal fibers.^ The role of histone H1 phosphorylation in PCC formation was investigated by acidurea polyacrylamide gel electrophoresis of total histone extracted from metaphase chromosomes and PCC following high frequency fusion. This investigation failed to demonstrate an extensive phosphorylation of H1 associated with PCC formation. However, significant dephosphorylation of superphosphorylated metaphase chromosome H1 was observed, indicating that interphase H1-phosphatase activity is dominant over metaphase H1 kinase activity. These observations provide evidence against models suggesting a role for H1 superphosphorylation in triggering mitotic condensation of chromosomes. ^

THE ULTRASTRUCTURAL ORGANIZATION OF THE HYPOGLOSSAL NUCLEUS IN THE RAT (SYNAPTOLOGY, CRANIAL NERVES)

An ultrastructural study of the hypoglossal nucleus (XII) in the rat has revealed two distinct neuronal populations. Hypoglossal motoneurons comprised the largest population of neurons in XII and were identified following injection of horseradish peroxidase (HRP) into the tongue. Motoneurons were large (25-50(mu)m), multipolar in shape and distributed throughout XII. The nucleus was large, round and centrally located, and the cytoplasm was characterized by dense lamellar arrays of rough endoplasmic reticulum. In contrast, a second population of small (10-18(mu)m), round to oval shaped neurons was found restricted to the ventral and dorsolateral regions of XII. The nucleus was markedly invaginated and eccentric, the cytoplasm scant and filled with free ribosomes, and the absence of lamellar arrays of rough endoplasmic reticulum was conspicuous. Neurons of this type were never found to contain HRP reaction product. These results demonstrate that the hypoglossal nucleus does not consist solely of motoneurons, but includes a distinctly separate, presumably non-motoneuronal pool. Arguments are presented in favor of this second neuron population being interneurons. The functional significance of these findings in relation to tongue control is discussed. ^

Alteration of CEACAM1 gene transcription in prostate cancer cells: The role of AR, Sp2, and HDAC

Cell to cell adhesion molecule (CEACAM1), a type II tumor suppressor, has been found to be down-regulated in prostate cancer cells. The mechanism that causes CEACAM1's down-regulation in tumorigenesis is unknown. Here we show that the transcriptional activity of CEACAM1 is decreased in prostate cancer cells. This decrease is not due to methylation of the CEACAM1's promoter, but rather to the alteration of transcription factors regulating CEACAM1 expression. ^ Since androgen/androgen receptors (AR) are potent regulators of prostate growth and differentiation, their role on CEACAM1 gene transcription was examined. The androgen receptor could directly increase CEACAM1 transcriptional activity in a ligand dependent manner by interacting with an AR consensus element that resides in the CEACAM1 promoter. However, AR binding to the CEACAM1 promoter is not related to the loss of CEACAM1 during prostate cancer progression. ^ Further analysis enabled us to determine the particular region in the CEACAM1 promoter that mediates a decrease in CEACAM1 transcriptional activity in prostate cancer cells. Upon further examination, we found that this CEACAM1 promoter region interacts with the Sp1, Sp2, and Sp3 transcription factors. However, only Sp2 expression was found to increase in prostate cancer cells. Inhibiting Sp2 from binding to the CEACAM1 promoter caused an increase in CEACAM1 transcriptional activity in prostate cancer cells. In addition, over-expressing Sp2 in normal prostate cells resulted in a decrease in CEACAM1 transcriptional activity and endogenous protein expression. These observations suggest that Sp2 is a transcription repressor of CEACAM1. Furthermore, prostate cancer cells treated with trichostatin A (TSA), a specific histone deacetylase (HDAC) inhibitor, activated CEACAM1 transcriptional activity. This implies that HDACs are involved in CEACAM1 transcriptional activity. Mutation of the Sp2 DNA binding region on the CEACAM1 promoter inhibited TSA activation of CEACAM1 transcriptional activity. This indicates that HDACs inhibit CEACAM1 transcriptional activity through Sp2. Base on these results, we propose that Sp2 is critical for down-regulating CEACAM1 expression, and one mechanism by which Sp2 represses CEACAM1 expression is by recruiting HDAC to the CEACAM1 promoter in prostate cancer cells. Collectively, these findings provide novel insights into mechanisms that cause the down-regulation of CEACAM1 expression in prostate cancer cells. ^