2005至2006组织化学与细胞生物学的研究概况

Histochem Cell Biol (2006) 126:743–788 DOI 10.1007/s00418-006-0253-7123REVIEWThe histochemistry and cell biology vade mecum: a review of 2005–2006Douglas J. Taatjes · Christian Zuber · Jürgen RothAccepted: 3 November 2006 / Published online: 24 November 2006© Springer-Verlag 2006Abstract The procurement of new knowledge and understanding in the ever expanding discipline of cell biology continues to advance at a breakneck pace. The progress in discerning the physiology of cells and tis-sues in health and disease has been driven to a large extent by the continued development of new probes and imaging techniques. The recent introduction of semi-conductor quantum dots as stable, speci W c mark-ers for both X uorescence light microscopy and electron microscopy, as well as a virtual treasure-trove of new X uorescent proteins, has in conjunction with newly introduced spectral imaging systems, opened vistas into the seemingly unlimited possibilities for experi-mental design. Although it oftentimes proves di Y cult to predict what the future will hold with respect to advances in disciplines such as cell biology and histo-chemistry, it is facile to look back on what has already occurred. In this spirit, this review will highlight some advancements made in these areas in the past 2years.Keywords Nucleus · Apoptosis · Stem cells · Angiogenesis · Extracellular matrixIntroductionThis year marks the centennial of the awarding of the Nobel Prize in Physiology or Medicine to the two iconic histologists, Camillo Golgi and Santiago Ramon y Cajal. They were recognized for their discoveries revealing the W ne structure of the nervous system,made possible by the selected impregnation of neurons with metallic silver. In the ensuing 100years, an astounding farrago of histochemical methods has been developed for cell and tissue examination at both the light and electron microscopic level. This review will summarize two year’s worth of multifarious articles highlighting a variety of histochemical techniques pub-lished in Histochemistry and Cell Biology . With a tip of the cap to Golgi and Cajal, we will embark on this review by commencing with studies investigating neu-ral tissues and cells.The Nobel Centennial: advanced imaging techniques in the study of neurosciencesMisgeld (2005) presented an excellent review on how modern in vivo imaging techniques can be applied to gain new information in studies of axon loss in development and disease, such as post-traumatic axon degeneration. Dubovy and colleagues (2006) used immuno X uorescence techniques to examine various basal lamina molecules in the dorsal root ganglia in the Wistar rat following constriction nerve injury. A uni-form appearance of laminin-1 staining was found in the naïve dorsal root ganglia, whereas in ligated nerves the laminin-1 immunostaining appeared more hetero-geneous around neuron-satellite glial cell units. TheD. J. Taatjes (&)Department of Pathology, Microscopy Imaging Center, College of Medicine, University of Vermont,Burlington, VT 05405, USAe-mail: douglas.taatjes@C. Zuber · J. RothDivision of Cell and Molecular Pathology,Department of Pathology, University of Zurich,8091 Zurich, Switzerlandlaminin-1 immunostaining corresponded with binding of the lectin Concanavalin-A and immunostaining for type IV collagen in both naïve and ligation-a V ected dorsal root ganglia. The results suggest that nerve liga-ture causes an altered content of basal laminae sur-rounding the bodies of primary sensory neurons and their satellite glial cell units.The expression of the leptin receptor (OB-R) was evaluated in rat dorsal root ganglion by immunohisto-chemistry, Western blot, and RT-PCR (Chen et al. 2006). Treatment of rats with 17 -estradiol resulted in an increased OB-R protein and mRNA in ovariecto-mized rat dorsal root ganglion, the e V ect of which could be ameliorated by tamoxifen. Moreover, OB-R colocalized 100% with ER , and about 15% with ER in dorsal root ganglion neurons. These results demon-strated that OB-R is expressed in rat dorsal root gan-glion, and that this expression may be regulated by estradiol in an ER -dependent pathway. To possess an in vitro tool for the investigation of spinal cord injury-induced degenerative and regenerative processes, Stav-ridis et al. (2005) prepared and characterized spinal cord slice cultures (SCSC) from Wistar rats.Akt, a main downstream molecule of PI3K cascade, is a crucial factor for cell survival and migration whose phosphorylation is blocked by PTEN (phosphatase and tensin). Tanriover and colleagues (2005) tested their hypothesis that spatial and temporal expression of PTEN is likely to modulate developing human brain cortical modeling by regulating Akt activation. They found evidence to suggest that Akt-mediated signaling plays an active role in cell migration, survival, and cere-bral cortical modeling during prenatal life and that PTEN is the most likely protein to regulate this signal-ing.Schwarzenbacher and co-workers (2005) investi-gated the appearance of odorant receptors in the olfac-tory epithelium of mice at various stages of prenatal development by immunohistochemistry and antibodies speci W c for di V erent odorant receptor subtypes. Odor-ant receptor proteins accumulated in dendritic knobs of OSNs before the initiation of ciliogenesis (embry-onic day 12) and the knob staining diminished when the W rst cilia were formed at embryonic day 13. During further stages of development until the formation of meshwork forming cilia, immunostaining for odorant receptors was detectable along the entire cilium. This group has now followed up on this work by identifying a novel population of olfactory marker protein (OMP)-expressing neurons found bilaterally in the anterior/ dorsal region of each nasal cavity at the septum of the mouse (Fleischer et al. 2006). The arrangement of the OMP-expressing cells is considerably altered during development: they appear at stage E13 and remain localized in the nasal epithelium during early stages; ganglion-shaped clusters are formed in the mesen-chyme during the perinatal phase, and by adulthood a W liform layer has formed beneath the nasal epithelium. These OMP cells proceed to form bundled long axonal processes projecting towards the brain, which may serve a distinct sensory function.Taste bud development in the chick was investigated by Ganchrow and colleagues (2006). In the chick, taste bud (gemmal) primordia appear on day E16, and immature spherical-shaped buds on E17. Injection of -bungarotoxin on E12 resulted in an absence of taste buds in the lower beak and palatal epithelium at days E17 and E21, although putative gemmal primordia remained. NCAM expression was not observed in oral epithelium, indicating that gemmal primordia are not nerve-dependent. Following unilateral geniculate gan-glion/otocyst excision on day E2.5, 12% of surviving ipsilateral geniculate ganglion cells sustained approxi-mately half of the unoperated gemmal counts at day E18 and day 1 post-hatch. Since the proportion of surviving buds following ganglion damage can be sustained at the same di V erentiated bud stage as on the operated side, these results suggest that taste bud maturation, maintenance and survival are nerve-dependent.Avendano and co-workers (2006) used injection with the anterograde tracers wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) and the Phaseolus vulgaris leucoagglutinin (PHA-L) to investigate whether the lateral intermediate (LI) and posterior (Po) groups of the thalamus also project to the striatum in the cat. Projections to the lateral part of the head and body of the caudate nucleus and to the putamen were found to arise from the ventral parts of the caudal subdivision of the LI in addition to the established sources in the intralaminar and ventral tha-lamic nuclei. From the medial division of the Po, few projections were observed to the putamen, and none to the caudal nucleus. Thalamostriatal W bers contained terminal and intercalated varicosities, suggesting that they form terminal and en passant synapses. The W bers from these thalamic sectors were unevenly distributed within the caudal nucleus, and corresponded to the acetylcholinesterase-poor striosomes and the extrastri-osomal matrix.BK channels are involved in controlling action potential duration and the W ring frequency and spike frequency adaptation for many neurons by leading to a fast after hyperpolarization after a neuronal action potential. Sausbier and colleagues (2006) produced an antibody against the 442 carboxy-terminal amino acids123of the BK channel -subunit, to study BK channel expression in the mouse. Using this antibody, they observed a high amount of immunoreactivity within the telencephalon in the cerebral cortex, olfactory bulb, basal ganglia and hippocampus. Lower levels of immunostaining were detected in the basal forebrain region and amygdala. Other regions expressing a high amount of BK channel staining were the nuclei of the ventral and dorsal thalamus and medial habenular nucleus, the fasciculus retro X exus and its termination in the mesencephalic interpeduncular nucleus, the periaquaeductal gray and raphe nuclei, the cerebellar cortex and locus coeruleus of the rhombencephalon, and the vestibular nuclei. At the cellular level, the BK channels display pre- and postsynaptic localizations. The mapping of the BK channel localization should help in the functional characterization of genetically manipulated mice.Treatment of animals with diethylstilbestrol (DES) can lead to debilitating e V ects. Shukuwa and col-leagues (2006) examined the e V ects of 20-day treat-ment with DES on follicle-stimulating hormone (FSH), luteinizing hormone (LH) and prolactin (PRL) cells in the pituitaries of male mice. The densities of FSH and LH cells were reduced, while that of PRL cells was increased following DES treatment. DES caused an increased expression of estrogen receptor (ER) in PRL cells. Cell proliferation assayed with an anti-Ki67 antibody was increased in some PRL cells 10–15days following DES treatment, and Pit-1 (pituitary-speci W c transcription factor)-positive cells were evident 5–15days post-treatment. Moreover, some FSH and LH cells became Pit-1 positive after 5–10days of treat-ment. Taken together, these results suggest that treat-ment of male mice with DES leads to an increase in PRL cells by inducing PRL cell proliferation, and by transdi V erentiation of FSH and LH cells into PRL cells.Much attention has been focused on neural innerva-tion of gastrointestinal tissues. In an excellent review, the discoverers Wörl and Neuhuber (2005) provide an update on the enteric co-innervation of motor end-plates in the esophagus with regard to its occurrence in a variety of species, including humans, its neurochemis-try, spatial relationships on motor endplates, ontogeny, and possible roles during esophageal peristalsis. Ewald and colleagues (2006) used confocal microscopy to determine colocalization patterns of vesicular gluta-mate transporter 1 (VGLUT1) with the intraganglionic laminar endings markers calretinin and VGLUT2 in rat esophagus. Additionally, VGLUT1 was observed in cholinergic and nitregic myenteric neuronal cell bod-ies, in W bers of the muscularis mucosae, and in esopha-geal endplates. Their results indicated that VGLUT1 immunoreactivity was present in both extrinsic and intrinsic innervation of the rat esophagus. In continua-tion of their previous studies (Ruan et al. 2004; Xiang and Burnstock 2004a, b) and others, Burnstock’s group (Xiang and Burnstock 2005) investigated by immuno-histochemistry the distribution of P2Y2 receptor-immunoreactive neurons and W bers in the guinea pig enteric nervous system, the most studied animal model in this area. They have extended these studies by investigating the distribution of P2Y6 and P2Y12 receptor-immunoreactive neurons and W bers and their relationship with calbindin, calretinin and nitric oxide (NO) synthase (Xiang and Burnstock 2006). They found that 30–36% of the ganglion cells in the myenteric plexus are P2Y6 receptor-immunoreactive neurons, with a wide distribution in the stomach, jejunum, ileum and colon. Approximately 28–35% of these neurons coexisted with NO synthase, and 41–47% with calretinin. Almost half of the ganglion cells in the myenteric and submucosal plexuses show P2Y12 receptor-immunoreactivity, which were also immunopositive for calbindin. Breh-mer and colleagues (2006) characterized human myen-teric neurons co-reactive for neuronal nitric oxide synthase (nNOS) and vasoactive intestinal peptide (VIP) for both their morphology and proportion with respect to the entire myenteric neuronal population. Using triple immunostaining for neuro W laments, VIP and nNOS, they identi W ed spiny neurons, which di V ered morphologically in the small intestine com-pared with the large. Neuronal tracing showed that 94% of the neurons ran anally while 6% ran orally from the myenteric plexus. The results indicated that human myenteric spiny neurons displaying both VIP and nNOS represent either inhibitory motor or descending interneurons. Also in humans, Furness and co-workers (2006) investigated by immunohistochem-istry the expression of protein kinase C (PKC) iso-forms in human intestine. PKC , PKC , and to some extent PKC were present in enteric neurons, while PKC was dominant in smooth muscle. Enteric glial cells expressed PKC and PKC , spindle-shaped cells in the mucosa stained for PKC and PKC , while simi-lar cells in the muscle layers stained for PKC and PKC . The presence of these PKC isoforms in the tis-sues was corroborated by Western blot analysis and RT-PCR. The results demonstrated the cell-type speci W c expression of PKC isoforms in intestinal enteric neurons and smooth muscle cells, and indicated that while the expression patterns are similar for both human and guinea pig tissues, some di V erences are found. Van Nassauw et al. (2005) provided evidence in favor of the hypothesis that NeuN, a neuronal nuclei123antibody, is a selective marker of intrinsic primary a V erent neurons in the guinea-pig gastrointestinal tract.Numerous other studies focussed on the develop-ment, and the functional and structural morphology of the brain vasculature, particularly the blood–brain bar-rier. Since an important structural component of the blood–brain barrier is the tight junction, Anstrom and colleagues (2006) used immunohistochemical tech-niques to determine if amounts of the tight junction proteins ZO-1, claudin, and occludin increase with gestational age in vessels of the germinal matrix and cortex in human premature infants. By 24weeks post-conception, tight junction proteins were present in both the germinal matrix and cortical vessels, but the amount of immunoreactivity was decreased in the ger-minal matrix of the youngest subjects. Moreover, also by 24weeks post-conception, the amount of tight junc-tion protein immunoreactivity was less in germinal matrix vessels as compared with cortical vessels, sug-gesting that tight junction maturation may progress along a super W cial to deep brain axis. These results indicate that di V erentiation of some functional special-izations is still undergoing progress in germinal matrix vessels during the third trimester of human fetal devel-opment.Orexin, a supposed neuromodulator, is expressed in neurons in the lateral hypothalamus which are impor-tant for the regulation of sleep and wakefulness, inges-tive behavior, and the coordination of these behaviors in an environmental context. To further identify down-stream e V ector targets of the orexin system, Berthoud et al. (2005) examined in detail orexin-A innervation of the three components of the caudal raphe nuclei in the medulla, known to harbor sympathetic preganglionic motor neurons involved in thermal, cardiovascular, and gastrointestinal regulation. Their W ndings sug-gested that orexin neurons in the hypothalamus can modulate brown fat thermogenesis, and cardiovascular as well as gastrointestinal functions by acting directly on neurons in the caudal raphe nuclei.Endogenous clocks, which control circadian rhythms are driven by the pivotal clock protein PER (Dunlap et al. 1999). Both circadian changes in PER content and its translocation from the cytoplasm to the nucleus appear to be crucial clock features. Zavodska et al. (2005) detected light-dependent PER-like proteins in a small set of cephalic ganglia of two insect species (W re-bat Thermobia domestica and cockroach Periplaneta americana). The behavior of nuclear PER-like proteins was opposite that of the cytoplasmic antigens that per-sist in constant darkness and disappeared in constant light. The sensitivity to photo regime suggested that both the nuclear and the cytoplasmic PER-like pro-teins are components of the biological clock.A cell line derived from a mouse brain tumor, the CAD cell line, when grown in a protein-free medium, di V erentiates to neuron-like cells with long cellular processes and expresses tyrosine hydroxylase (TH) and certain neuron-speci W c proteins. CAD cells also express various neuropeptides, chromogranins, GAP-43 and GFAP (Li et al. 2005). Thus, CAD cells may represent a good model for studying intracellular transport and the in X uence of di V erent trophic/ growth factors on the expression of various neuronal characteristics.Protein 4.1G is a commonly expressed cytoskeletal protein that was also detectable in mature columnar epithelial cells of normal rat intestine (Ohno et al. 2004). In another study, Ohno et al. (2005) concen-trated on the distribution of protein 4.1G in the central nervous system of mice. Interestingly, protein 4.1G-positive cells corresponded to microglia, with other glial cells and neuronal cells being unreactive.A most intriguing distribution of the vanilloid recep-tor type 1 (TRPV1) was recently reported by Faus-sone-Pellegrini and coworkers (2005). In addition to its presence in neurons and nerve W bers of the human stomach, TRPV1-immunoreactivity was also found in the parietal cells. Because of the presence of TRPV1 in both nerve structures and parietal cells, the possibility of a combined e V ect on hydrochloric acid secretion was suggested. Tsukagoshi and colleagues (2006) examined the expression of TRPV1 and TRPV2 in calcitonin gene-related peptide (CGRP) positive sensory neurons projecting to the skin and skeletal muscles of the rat dorsum. Of the small CGRP(+) neurons projecting to the skin, 54% were positive for TRPV1, while 12% were positive for TRPV2. Likewise, of the small CGRP(+) neurons projecting to skeletal muscle, 53% were positive for TRPV1 and 8% for TRPV2. With respect to the skeletal muscle, more CGRP(+) neurons projected to the trapezius muscle versus the longissi-mus. Finally, the colocalization percentage of CGRP and TRPV1 on the sensory nerves was greater in the trapezius than in the longissimus and the skin.Brouns and colleagues (2006) examined the loca-tion, morphology, and neurochemical coding of sub-epithelial receptor-like structures that have been previously reported in the wall of large diameter air-ways of the lung. They compared their observations with those obtained for pulmonary neuroepithelial bodies (NEBs), complex intraepithelial sensory airway receptors. They found branching laminar subepithelial receptor-like endings intercalated in the smooth mus-cle cell layer of rat conducting airways. They referred123to these receptors as smooth muscle-associated airway receptors (SMARs). The neurochemical coding and receptor-like appearance of SMARs was very reminis-cent of NEBs. Due to the expression of markers for mechanoreceptor terminals in other organs, the authors postulate that both SMARs and the vagal nodose nerve terminals in NEBs may represent the morphological counterparts of at least subsets of the population of physiologically characterized myelinated vagal airway mechanoreceptors. Also within the lung, D’Angelis and colleagues (2006) examined the expression of C-type natriuretic peptide (CNP) and natriuretic peptide receptor B (NPR-B) in pulmonary epithelium of the developing fetal lamb and following transition to breathing. CNP acts through NPR-B to increase intra-cellular cGMP and can a V ect the movement of ions across the pulmonary epithelium. At 100days of gesta-tion, no staining for either CNP or NPR-B was found in the pulmonary epithelium, whereas by 136days of gestation, prominent staining for both molecules was found within type II alveolar cells, Clara cells and cili-ated epithelium of the upper airways. By three and four days following birth, staining was not found in type II cells or ciliated epithelial cells, and only at a reduced level in Clara cells. These results suggest that CNP may contribute to the maintenance of the X uid-W lled lung through the regulation of trans-epithelial ion X ux.The nucleus and its dynamic compartmentsThe nucleus has been the focus of intense research regarding its structural organization, functional com-partmentalization and the dynamics of its substructures (Fakan 2004; Misteli 2005; Olson and Dundr 2005; Roix and Misteli 2002). Recent reviews have focused on various structure-function aspects and dynamics of de W ned components of the nucleolus (Hernandez-Ver-dun 2006a, b). For instance, time-lapse video micros-copy and X uorescent recovery after photobleaching (FRAP) demonstrated that the mobility of GFP-tagged nucleolar proteins was slower in the nucleolus than in the nucleoplasm. Evidence was presented that the nucleolus appears at the center of several tra Y ck-ing pathways in the nucleus. Furthermore, aspects of the nucleolus and diseases as well as of nucleolar functions di V erent from ribosome biogenesis were reviewed. In another review, Görisch et al. (2005) con-centrate on mobile subnuclear organelles involved in RNA processing, transcriptional regulation and antivi-ral defense such as Cajal and promyelocytic leukemia (PML) bodies.Baran et al. (2006) have studied the localization of the nuclear DNA helicase II (NDH II), which is the bovine homolog of human RNA helicase A, in somatic cells and oocytes. The focus of their studies was on the dynamic changes in the redistribution of NDH II dur-ing the growth phase of the bovine oocytes. In W bro-blasts, W ne granular staining of NDH II was present throughout the nucleoplasm, excluding the reticulated nucleoli. In contrast, in growing oocytes isolated from early antral follicles, the large reticulated nucleoli exhibited strong positivity for NDH II together with immunostaining of upstream binding factor (UBF) and RNA polymerase I subunit (PAF53). When oocyte growth was terminated, NDH II was preferentially located at the nucleolar periphery together with pro-teins of W brillar centers. In fully grown oocytes, NDH II was still present in the thin periphery shell around the compact nucleolar core. Semiquantitative RT-PCR revealed that the average signal of NDH II mRNA in fully grown oocytes was only 40% of that of growing oocytes. This was con W rmed by Western blot analysis. Collectively, this demonstrates a signi W cant decrease in NDH II gene expression and in NDH II mRNA trans-lation in correlation with termination of oocyte growth, and that NDH II expression parallels the activity of ribosomal RNA biosynthesis in growing bovine oocytes.Smetana et al. (2006a) studied early leukemic granu-locytic and plasmacytic precursors in vitro and in vivo to gain information on the intranucleolar distribution of AgNORs (silver stained nucleolus organizer regions). In most cells, AgNORs appeared as clusters in the whole nucleolar body. However, in some leukemic early granulocytic precursors, i.e., in myeloblasts and promyelocytes enlarged AgNORs were translocated in the nucleolar peripheral part and the number of trans-located AgNORs at the nucleolar periphery was sig-ni W cantly smaller. This phenomenon could be produced by experimental aging, i.e., starving of cul-tured leukemic early granulocytic precursors (HL-60 and K562 cells) in vitro and seems to be reversible. It was proposed that the translocation of the reduced number of AgNORs to the nucleolar periphery in some blastic leukemic hematopoietic cells might be a useful marker of aging at the single cell level. The nucleolar structure and the activity of NopA100, a nucleolin-like protein, during the cell cycle in prolifer-ating plant cells was analyzed by Gonzalez-Camacho and Medina (2006). A relationship between the increase in nucleolar activity, cell-cycle progression, nucleolar structure, the activity of NopA100, and the proteolysis of this nucleolin-like protein was found. Diez et al. (2006) report the presence in the nucleolus of a reverse123transcriptase previously found to be associated with telomeres in Chironomus. By immuno X uorescence, a distinct pattern of staining in the giant nucleoli of polytenized cells was observed, consisting of a de W nite necklace pattern that did not completely colocalize with W brillarin or nucleolin and appeared to be di V er-ent from that of typical nucleolar components. These results indicate that the putative Chironomus telomere elongation machinery and telomerase share a nucleolar localization and reinforce the idea that nucleoli are functionally linked to telomere maintenance.Cajal and promyelocytic leukemia bodies are dis-crete nuclear structures with speci W c molecular signa-tures. RNA polymerase II and many transcription factors have been identi W ed within these compart-ments. Xie and Pombo (2006) have examined di V erent phosphorylated forms of polymerase II and newly made RNA in Cajal and PML bodies using high-reso-lution imaging of ultrathin cryosections. They show that Cajal bodies contain polymerase II phosphory-lated on Ser5, and not the Ser2-phosphorylated (active) form on newly made RNA. Hence, the pres-ence of polymerase II in the absence of transcriptional activity suggests that Cajal bodies have roles in poly-merase assembly or transport, but not in gene tran-scription. PML bodies did not contain polymerase II or nascent RNA in HeLa cells, at the resolution achieved by electron microscopy, but are often surrounded by these markers at distances >25nm. It was concluded that although PML bodies are present in transcription-ally active areas of the nucleus, they are not generally sites of polymerase II assembly, transport or activity.The sites of DNA synthesis in HeLa cell nucleus were studied by immunoelectron microscopic detec-tion of incorporated biotin-dUTP (Philimonenko et al. 2006). Two types of DNA synthetic sites/replication factories were found: (1) electron-dense structures–replication bodies (RB), and (2) focal replication sites with no distinct underlying structure-replication foci (RF). By double immunogold labeling both structures contain (a) proteins involved in DNA replication (DNA polymerase alpha, PCNA), (b) regulators of the cell cycle (cyclin A, cdk2), and (c) RNA processing components like Sm and SS-B/La auto antigens, p80-coilin, hnRNPs A1 and C1/C2. However, at least four regulatory and structural proteins (Cdk1, cyclin B1, PML and lamin B1) di V er in their presence in RB and RF. Moreover, in contrast to RF, RB have structural organization. These data suggest possible di V erences in genome regions replicated by these two types of repli-cation factories.Mathee et al. (2006) analyzed the nanostructure of speci W c chromatin regions and nuclear complexes. For this, they applied spatially modulated illumination (SMI) microscopy, which is a method of wide W eld X uo-rescence microscopy featuring interferometric illumi-nation. SMI delivers structural information about nanoscale features in X uorescently labeled cells and structural changes in the context of gene activation and chromatin remodeling may be revealed. Speci W cally, SMI microscopy was used to size measurements of the 7q22 gene region, giving a size estimate of 105§16nm which corresponds to an average compaction ratio of 1:324. The absence of a correlation between the mea-sured and genomic sizes of the various gene regions indicate that a high variability in chromatin folding is present, with factors other than the sequence length contributing to the chromatin compaction.The rapid exchange of the glucocorticoid receptor (GR) at the mouse mammary tumor virus (MMTV) promoter depends on chaperone and proteasome activity, and may also involve chromatin remodeling activity. The latter possibility was studied by Stavreva and McNally (2006) using photobleaching technology in live cells. Inhibition of H1 phosphorylation, chroma-tin remodeling and transcription from MMTV was accomplished by long-term blocking of Cdk2 protein kinase activity whereby Cdk2 was recruited by a tandem array of MMTV promoters. Furthermore, following a brief Cdk2 inhibition by a selective cyclin-dependent kinase inhibitor (Roscovitine), transcrip-tion from MMTV drops and GR exchange at MMTV became slower, with a fraction of GR molecules now tightly bound at the promoter. This immobile fraction is absent elsewhere in the nucleus, suggesting a speci W c e V ect of Cdk2 inhibition on GR-MMTV interactions. These are the W rst live cell data suggesting a role for H1 phosphorylation, and by implication chromatin remod-eling, in rapid exchange of GR at MMTV.Brink et al. (2006) showed that truncated hetero-chromatin protein 1 (HP1) lacking a functional chromodomain (CD) induces heterochromatinization upon in vivo targeting. At the single cell level it was analyzed whether HP1 lacking its functional CD is able to induce heterochromatinization in vivo. A lac-operator array-based system was used to target EGFP-lac repressor tagged truncated HP1alpha and HP1beta to a lac operator containing gene-ampli W ed chromosome region in living cells. It could be shown that CD-less HP1alpha can induce chromatin conden-sation, whereas the e V ect of truncated HP1beta is less pronounced. Thus, after lac repressor-mediated tar-geting, HP1alpha and HP1beta without a functional CD are able to induce heterochromatinization. The replication of centromeric heterochromatin in space and time in mouse W broblasts was analyzed during S123。