Wilhelm bernhard workshop on the cell nucleus

LOCALIZATION OF COMPONENTS OF THE SIGNAL RECOGNITION PARTICLE IN THE AMPLIFIED NUCLEOLI OF XENOPUS OOCYTES

Sommerville J.¹, Mullin A.¹, Gilchrist K.¹, Lewandowski L.B.², Brumwell C.L.², Politz J.C.² and Pederson T.².

¹School of Biology, Bute Medical Buildings, University of St Andrews, St Andrews, Scotland; ²Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, USA.
The nucleolus is the site of pre-ribosome assembly, but this compartment of the nucleus is now known to have other functions. One additional function came from the discovery that the signal recognition particle (SRP) RNA traffics through the nucleolus. Here we report the use of Xenopus oocytes to track the incorporation of both SRPRNA and a protein component of the SRP into the large, amplified, extrachromosomal nucleoli of germinal vesicles (oocyte nuclei). GFP-tagged SRP19 protein injected into the oocyte cytoplasm was imported into the nucleus and rapidly localized in the nucleoli. The dynamics of incorporation revealed that at 2 h post-injection GFP-SRP19 was located mainly in the dense fibrillar component (DFC) of the nucleoli and by 4 h the signal had spread throughout the granular component (GC). In contrast, Alexa 488-tagged SRPRNA injected into the cytoplasm was not imported into the nucleus, but on injection directly into the nucleus it was targeted to the nucleoli. That SRPRNA is normally present in extrachromosomal nucleoli was confirmed by in situ hybridization with complementary Cy3-labelled probes. Treatment of injected oocytes with leptomycin B, a specific inhibitor of the CRM1 (exportin-1) nuclear export pathway, caused GFP-SRP19 protein to accumulate in the nucleoplasm, suggesting that the SRP uses the same nuclear export pathway as does the 60S ribosomal subunit. These results establish that extrachromosomal nucleoli retain the same affinity for SRP components as is observed with somatic cell nucleoli that are built around the chromosomal nucleolar organizer. This suggests that the developmental strategy of amplifying nucleoli during amphibian oogenesis may be to produce a maternal stockpile not only of ribosomes, as classically envisioned, but also of signal recognition particles.

ROLE OF THE NUCLEOLUS IN HUMAN REPRODUCTION

Kittur N.¹, Bazett-Jones D.P² and Meier U.T. ¹

¹Department of Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York, USA; ²The Hospital For Sick Cbildren, University of Toronto, Ontario, Canada
The ultrastructurally well-defined nucleolar channel system (NCS) consists of intranuclear stacks of membrane cisternae embedded in an electron dense matrix. It is specific to the buman endometrial epithelium where it appears transiently post-ovulation, coincident with the height of receptivity. Molecularly, the NCS is uncharacterized. By overexpression of the nucleolar chaperone Noppl40 we induced intranuclear membrane systems (R-rings) in tissue culture cells that are indistinguishable from the NCS. Using histo-chemical labeling for the endoplasmic reticulum (ER) marker enzyme glucose-6-phosphatase, we show that the NCS, like the R-rings, consists of bona fide ER. Not only is this the first labeling of the NCS but it also suggests molecular identity of NCS and R-rings. Noppl40 localizes to the R-rings and apparently the NCS implicating this protein as a key player in NCS/R-ring formation Indeed, we provide evidence that NCS/ring biogenesis is initiated by a calcium-mediated Nopp140-membrane interaction. Thus, Noppl40 binds calcium and complexed calcium is enriched between the membrane stacks of the NCS/R-rings as determined by electron spectroscopic imaging. Moreover, calcium chelators delay R-ring formation. A direct role for the NCS in human reproduction is supported by our observation that the NCS occurs in and adjacent to cells exhibiting pinopodes, apical membrane protrusions thought to be involved in blastocyst attachment. Specifically, we speculate that the NCS affects the localization and/or expression of integral membrane proteins (e.g., by sequestering them within the nucleus) and thereby locally creating an epithelial cell surface that is favorable for blastocyst attachment. Indeed, CLIMP 63 and p58/ERGIC53, two integral membrane proteins, are misrouted to the R-rings. More importantly, expression of the plasma membrane marker, caveolin-1, is reduced on the surface of R-ring positive cells. Therefore, in the special case of human endometrium, the omnipresent nucleolus or some of its components may be crucial for reproduction.

CELL DEATH & NUCLEAR MARKERS OF DISEASE

FURTHER CONSIDERATIONS ON APOPTOTIC CELL NUCLEAR DOMAINS.

¹Burattini S., ¹Luchetti F., ¹Battistelli M., ^1,2Falcieri E.

¹Istituto di Scienze Morfologiche, University of Urbino “Carlo Bo”, and ²ITOI-CNR, Istituti Ortopedici Rizzoli, Bologna, Italy
Interphase nuclear chromatin is generally organized in particular and widely known domains which appear relatively comparable in various cell types, as well as after different technical procedures. Differently, apoptotic death induces a deep, presumably irreversible nuclear rearrangement, which extensively involves chromatin, and, even if less evidently, most other nuclear components. In this work we compare cell lineages known to be very different both in biological and morphological patterns, triggered to apoptosis by means of various physical and chemical agents. HL60 (promyelocytic), U937 (monoblastoid) and K562 (from human chronic myeloid leukemia) have been chosen as myeloid models. Thymocytes, CESS (B lymphoblasts) Jurkat and Molt-4 (T lymphoblasts) have been analysed as lymphoid cells (Mariani et al., 1999; Falcieri et al., 2000a). Hyperthermia, UV and gamma radiations were utilized as physical apoptotic triggers, while staurosporine, camptothecin, etoposide, methotrexate, interferon, mimosine and, very recently, a number of triazoles were used as chemical inducers. Apoptosis has been also investigated in a megakarioblastic model during platelet differentiation (Falcieri et al., 2000b) and, finally, in erythropoietin-related erythroid maturation of CD34 precursors. Nuclear changes were studied by light, fluorescence and electron microscopy, as well as by means of a number of cytochemical approaches (Luchetti et al., 2002). All these observations demonstrate that the behavior of apoptotic nuclear domains takes place in a higlily regulated and widely reproducible way, relatively independently of cell system and biochemistry.

References:

Mariani A.R. et al., Anat Rec, 254:1, 1999

Falcieri E. et al., Histochem Cell Biol, 113:135, 2000a

Falcieri E. et al., Anat Rec, 253:90, 2000b

Luchetti F. et al., Apoptosis 7:143, 2002


NUCLEAR CHANGES IN BREAST CANCER CELLS DURING EARLY PHASES OF CELL DEATH AND CELL GROWTH.

Losa G.A.^1,2, Castelli C.¹, Nonnenmacher T.F. <sup>1,3

1</sup>Institute of Scientific Interdisciplinary Studies, Locarno, and ²Faculty of Sciences, University of Lausanne, Switzerland; ³Department of Mathematical Physics, University of Ulm, Germany.
Fractal morphometry and grey level co-occurrence matrix analysis (GLCM) were applied to investigate early phases of cell growth and cell death in human breast cancer cells. Changes in the ultrastructural complexity of external perinuclear membrane (ENM), eu- and heterochromatins and in the textural features of selected nuclear domains (ROIs) were assessed in estrogen-insensitive SK-BR-3 breast cancer cells induced to apoptosis by 1 μM calcimycin, an apoptogenic Ca²⁺ ionophore. The reorganization of ENM and nuclear membrane-bound heterochromatin (NMBHC) was also established in estrogen-sensitive MCF-7 breast cancer cells triggered by 17-ß-estradiol and glucocorticoid dexamethasone. SK-BR-3 cells entered the early stage of apoptosis after 12-24 h of calcimycin treatment and had fractal dimension (FD) values of perinuclear membranes lower than in untreated cells, indicating a loss of morphological complexity. Changes of the chromatin texture within the entire nucleus and in ROIs were even more pronounced in treated cells as evaluated by decreased fractal dimensions and GLCM textural parameters. At the opposite, soon after a short treatment (five minutes) with 17-beta-estradiol, MCF-7 cells displayed an enhanced ultrastructural irregularity of NMBHC (higher FD values) while reduced with dexamethasone (lower FD) when compared to NMBHC from untreated cells. Unexpectedly, neither steroid modified ENM ultrastructure in these cells. Both fractal and GLCM analyses confirmed that the nuclear morphological reorganization imputable to a loss of structural complexity occurred in the early stage of apoptosis of SK-BR-3 cells while an increase was measured at the beginning of proliferation in estrogen-sensitive MCF-7 cells. These morphological-ultrastructural traits within the entire nucleus and in selected nuclear domains occurred well before the detection of conventional cellular markers and were accompanied by enzymatic changes at the cell periphery.

MODELING APOPTOTIC CHROMATIN CONDENSATION IN NUCLEI ISOLATED FROM NORMAL CELLS

Widlak P.

Department of Experimental and Clinical Radiobiology, Center of Oncology, Gliwice, Poland
Hallmarks of the terminal stages of apoptosis are genomic DNA fragmentation and chromatin condensation. Nuclease primarily responsible for apoptotic oligonucleosomal DNA fragmentation is DFF40 (DNA Fragmentation Factor 40 kD), also termed CAD (Caspase-activated DNase). DFF40/CAD triggers one of the pathways of apoptotic chromatin condensation. Another pathway of apoptotic chromatin condensation is triggered by mitochondrial AIF (Apoptosis-inducing Factor), which induce large-scale DNA fragmentation possibly activating other nucleases. Proteolysis of nuclear proteins, like lamin A cleavage by caspase-6, is required for complete breakdown of nuclei in cells undergoing apoptosis. Here, the mechanism of chromatin condensation triggered by DFF40/CAD has been studied. We have found that DNA fragmentation per se of isolated nuclei from non-apoptotic cells induces chromatin condensation under physiological ionic strengths that closely resembles the morphology seen in apoptotic cells. This phenomenon is not specific for an apoptotic nuclease DFF40/CAD. Although there is no energy requirement for this process, such condensation can be inhibited without affecting DNA fragmentation by two different pretreatment of nuclei: (i) exposing to reagents that bind to the DNA minor groove, which disrupt native nucleosomal DNA wrapping, and (ii) stabilizing internal nuclear components. Interestingly, inhibition of nuclear F-actin depolymerization or promoting its formation also reduces chromatin condensation. Therefore, the ability of chromatin fragments with native nucleosomal DNA wrapping to form large clumps of condensed chromatin during apoptosis requires the apparent disassembly of internal nuclear structures that may normally constrain chromosome subdomains in non-apoptotic cells. Consistently, chromatin condensation triggered by DNA fragmentation was greatly enhanced when isolated nuclei were first incubated with caspase-3 and caspase-6. In conclusion, morphological changes that closely resembles apoptotic chromatin condensation in vivo can be induced by well defined factors in purified normal cell nuclei. Such factors include nucleases that fragmentize chromatin and proteases that cleave nuclear proteins and enhance intranuclear mobility of chromatin fragments.

SYNCHRONIZED ONSET OF NUCLEAR AND SURFACE CHANGES DURING APOPTOSIS OF HUMAN, RAT AND MOUSE LYMPHOCYTES

Dini L. and Abbro L.

Department. of Biological and Environmental Science and Technology, University of Lecce, Italy