Relationships between the structural and functional organization of the turtle cell nucleolus.

The nucleolus is a multifunctional structure of the eukaryotic cell nucleus. However, its primary role is ribosome formation. Although the factors and mechanisms involved in ribogenesis are well conserved in eukaryotes, two types of nucleoli have been observed under the electron microscope: a tricompartmentalized nucleolus in amniotes and a bicompartmentalized nucleolus in other species. A recent study has also revealed that turtles, although belonging to amniotes, displayed a nucleolus with bipartite organization, suggesting that this reptile group may have carried out a reversion phenomenon during evolution. In this study, we examine in great detail the functional organization of the turtle nucleolus. In liver and spleen cells cultured in vitro, we confirm that the turtle nucleolus is mainly formed by two components: a fibrillar zone surrounded by a granular zone. We further show that the fibrillar zone includes densely-contrasted strands, which are positive after silver-stained Nucleolar Organizer Region (Ag-NOR) staining and DNA labelling. We also reveal that the dense strands condensed into a very compact mass within the fibrillar zone after a treatment with actinomycin D or 5,6-dichlorobenzimidazole riboside. Finally, by using pulse-chase experiments with BrUTP, three-dimensional image reconstructions of confocal optical sections, and electron microscopy analysis of ultrathin sections, we show that the topological and spatial dynamics of rRNA within the nucleolus extend from upstream binding factor (UBF)-positive sites in the fibrillar zone to the granular zone, without ever releasing the positive sites for the UBF. Together, these results seem to clearly indicate that the compartmentalization of the turtle nucleolus into two main components reflects a less orderly organization of ribosome formation.

Electron tomography reveals changes in spatial distribution of UBTF1 and UBTF2 isoforms within nucleolar components during rRNA synthesis inhibition.

Upstream binding transcription factor (UBTF) is a co-regulator of RNA polymerase I by constituting an initiation complex on rRNA genes. UBTF plays a role in rDNA bending and its maintenance in "open" state. It exists as two splicing variants, UBTF1 and UBTF2, which cannot be discerned with antibodies raised against UBTF. We investigated the ultrastructural localization of each variant in cells synthesizing GFP-tagged UBTF1 or UBTF2 by using anti-GFP antibodies and pre-embedding nanogold strategy. Detailed 3D distribution of UBTF1 and 2 was also studied by electron tomography. In control cells, the two isoforms are very abundant within fibrillar centers, but their repartition strongly differs. Electron tomography shows that UBTF1 is disposed as fibrils that are folded in coils whereas UBTF2 is localized homogenously, preferentially at their cortical area. As UBTF is a useful marker to trace rDNA genes, we used these data to improve our previous model of 3D organization of active transcribing rDNA gene within fibrillar centers. Finally, when rRNA synthesis is inhibited during actinomycin D treatment or entry in mitosis, UBTF1 and UBTF2 show a similar distribution along extended 3D loop-like structures. Altogether these data suggest new roles for UBTF1 and UBTF2 isoforms in the organization of active and inactive rDNA genes.

Various Nucleolar Stress Inducers Result in Highly Distinct Changes in Water, Dry Mass and Elemental Content in Cancerous Cell Compartments: Investigation Using a Nano-Analytical Approach.

Rationale: Numerous chemotherapeutic drugs that affect ribosome biogenesis in the nucleolus induce nucleolar stress. Improving our understanding of the effects of these drugs will require uncovering and comparing their impact on several biophysical parameters of the major cell compartments. Here, we quantified the water content and dry mass of cancerous cells treated with CX-5461, DRB or DAM to calculate macromolecular crowding and the volume occupied by free water, as well as elemental content. Methods: HeLa-H2B-GFP cells were treated with CX-5461, DRB or DAM. Water content and dry mass were measured in numerous regions of interest of ultrathin cryo-sections by quantitative scanning transmission electron microscope dark-field imaging and the elements quantified by energy dispersive X-ray spectrometry. The data were used to calculate macromolecular crowding and the volume occupied by free water in all cell compartments of control and treated cells. Hydrophobic and unfolded proteins were revealed by 8-Anilinonaphtalene-1-sulfonic acid (ANS) staining and imaging by two-photon microscopy. Immunolabeling of UBF, pNBS1 and pNF-κB was carried out and the images acquired with a confocal microscope for 3D imaging to address whether the localization of these proteins changes in treated cells. Results: Treatment with CX-5461, DRB or DAM induced completely different changes in macromolecular crowding and elemental content. Macromolecular crowding and elemental content were much higher in CX-5461-treated, moderately higher in DRB-treated, and much lower in DAM-treated cells than control cells. None of the drugs alone induced nucleolar ANS staining but it was induced by heat-shock of control cells and cells previously treated with DAM. UBF and pNBS1 were systematically co-localized in the nucleolus of CX-5461- and DAM-treated cells. pNF-κB only localized to the nucleolar caps of pre-apoptotic DAM-treated cells. Conclusion: We directly quantified water and ion content in cell compartments using cryo-correlative electron microscopy. We show that different chemotherapeutic nucleolar stress inducers result in distinctive, thus far-unrecognized changes in macromolecular crowding and elemental content which are known to modify cell metabolism. Moreover we were able to correlate these changes to the sensitivity of treated cells to heat-shock and the behavior of nucleolar pNBS1 and pNF-κB.

Nucleolar sub-compartments in motion during rRNA synthesis inhibition: Contraction of nucleolar condensed chromatin and gathering of fibrillar centers are concomitant.

The nucleolus produces the large polycistronic transcript (47S precursor) containing the 18S, 5.8S and 28S rRNA sequences and hosts most of the nuclear steps of pre-rRNA processing. Among numerous components it contains condensed chromatin and active rRNA genes which adopt a more accessible conformation. For this reason, it is a paradigm of chromosome territory organization. Active rRNA genes are clustered within several fibrillar centers (FCs), in which they are maintained in an open configuration by Upstream Binding Factor (UBF) molecules. Here, we used the reproducible reorganization of nucleolar components induced by the inhibition of rRNA synthesis by Actinomycin D (AMD) to address the steps of the spatiotemporal reorganization of FCs and nucleolar condensed chromatin. To reach that goal, we used two complementary approaches: i) time-lapse confocal imaging of cells expressing one or several GFP-tagged proteins (fibrillarin, UBF, histone H2B) and ii) ultrastructural identification of nucleolar components involved in the reorganization. Data obtained by time lapse confocal microscopy were analyzed through detailed 3D imaging. This allowed us to demonstrate that AMD treatment induces no fusion and no change in the relative position of the different nucleoli contained in one nucleus. In contrast, for each nucleolus, we observed step by step gathering and fusion of both FCs and nucleolar condensed chromatin. To analyze the reorganization of FCs and condensed chromatin at a higher resolution, we performed correlative light and electron microscopy electron microscopy (CLEM) imaging of the same cells. We demonstrated that threads of intranucleolar condensed chromatin are localized in a complex 3D network of vacuoles. Upon AMD treatment, these structures coalesce before migrating toward the perinucleolar condensed chromatin, to which they finally fuse. During their migration, FCs, which are all linked to ICC, are pulled by the latter to gather as caps disposed at the periphery of nucleoli.

Stage-Specific Changes in the Water, Na+, Cl- and K+ Contents of Organelles during Apoptosis, Demonstrated by a Targeted Cryo Correlative Analytical Approach.

Many studies have demonstrated changes in the levels of several ions during apoptosis, but a few recent studies have reported conflicting results concerning the changes in water content in apoptotic cells. We used a correlative light and cryo-scanning transmission electron microscopy method to quantify water and ion/element contents simultaneously at a nanoscale resolution in the various compartments of cells, from the onset to the end of apoptosis. We used stably transfected HeLa cells producing H2B-GFP to identify the stages of apoptosis in cells and for a targeted elemental analysis within condensed chromatin, nucleoplasm, mitochondria and the cytosol. We found that the compartments of apoptotic cells contained, on average, 10% more water than control cells. During mitochondrial outer membrane permeabilization, we observed a strong increase in the Na+ and Cl- contents of the mitochondria and a strong decrease in mitochondrial K+ content. During the first step in apoptotic volume decrease (AVD), Na+ and Cl- levels decreased in all cell compartments, but remained higher than those in control cells. Conversely, during the second step of AVD, Na+ and Cl- levels increased considerably in the nucleus and mitochondria. During these two steps of AVD, K+ content decreased steadily in all cell compartments. We also determined in vivo ion status during caspase-3 activity and chromatin condensation. Finally, we found that actinomycin D-tolerant cells had water and K+ contents similar to those of cells entering apoptosis but lower Na+ and Cl- contents than both cells entering apoptosis and control cells.

The elastin peptide (VGVAPG)3 induces the 3D reorganisation of PML-NBs and SC35 speckles architecture, and accelerates proliferation of fibroblasts and melanoma cells.

During melanoma tumour growth, cancerous cells are exposed to the immediate surrounding the micro- and macro environment, which is largely modified through the degradation of the extracellular matrix by fibroblast-derived metalloproteinases. Among the degradation products, (VGVAPG)3, an elastin peptide is known to stimulate the proliferation of both fibroblasts and cancerous cells by binding to the elastin-binding receptor and activating the MEK/ERK signal transduction pathway. As this process strongly modifies mRNA synthesis, we investigated its effect on the relative three-dimensional organisation of the major partners of the mRNA splicing machinery: promyelocytic nuclear bodies (PML-NBs ) and splicing component 35 speckles (SC35) of normal fibroblasts and melanoma SK-MEL-28 cells. SC35 and PML-NBs proteins were immunolabeled and imaged by confocal microscopy within these cells cultured with (VGVAPG)3. Three-dimensional reconstruction was performed to elucidate the organisation of PML-NBs and SC35 speckles and their spatial relationship. In G0 cells, SC35 speckles were sequestered in PML-NBs. Shortly after (VGVAPG)3 stimulation, the three-dimensional organisation of PML-NBs and SC35 speckles changed markedly. In particular, SC35 speckles gradually enlarged and adopted a heterogeneous organisation, intermingled with PML-NBs. Conversely, inhibition of the elastin-binding protein or MEK/ERK pathway induced a remarkable early sequestration of condensed SC35 speckles in PML-NBs, the hallmark of splicing inhibition. The 3D architecture of speckles/PML-NBs highlights the modulation in their spatial relationship, the multiple roles of PML-NBs in activation, inhibition and sequestration, and provides the first demonstration of the dependence of PML-NBs and SC35 speckles on the elastin peptide for these functions.

Targeted nano analysis of water and ions in the nucleus using cryo-correlative microscopy.

The cell nucleus is a crowded volume in which the concentration of macromolecules is high. These macromolecules sequester most of the water molecules and ions which, together, are very important for stabilization and folding of proteins and nucleic acids. To better understand how the localization and quantity of water and ions vary with nuclear activity, it is necessary to study them simultaneously by using newly developed cell imaging approaches. Some years ago, we showed that dark-field cryo-Scanning Transmission Electron Microscopy (cryo-STEM) allows quantification of the mass percentages of water, dry matter, and elements (among which are ions) in freeze-dried ultrathin sections. To overcome the difficulty of clearly identifying nuclear subcompartments imaged by STEM in ultrathin cryo-sections, we developed a new cryo correlative light and STEM imaging procedure. This combines fluorescence imaging of nuclear GFP-tagged proteins to identify, within cryo ultrathin sections, regions of interest which are then analyzed by STEM for quantification of water and identification and quantification of ions. In this chapter we describe the new setup we have developed to perform this cryo-correlative light and STEM imaging approach, which allows a targeted nano analysis of water and ions in nuclear compartments.

Encapsulated Ruthenium(II) Complexes in Biocompatible Poly(d,l-lactide-co-glycolide) Nanoparticles for Application in Photodynamic Therapy.

The elaboration, characterisation and efficiency of potential two-photon-excited photodynamic therapy (PDT) treatment of new poly(d,l-lactide-co-glycolide) nanoparticles loaded with ruthenium(II) complexs are presented. The materials are based on the encapsulation of RuII complexes through an all-biocompatible process. The size of the nanoparticles is around 100 nm. The internal concentration is several orders of magnitude higher than the overall concentration, which leads to a more efficient and targeted effect. The therapeutic potential for PDT of these nanoparticles has been studied in vitro on C6 glioma cells.

Changes to cellular water and element content induced by nucleolar stress: investigation by a cryo-correlative nano-imaging approach.

The cell is a crowded volume, with estimated mean mass percentage of macromolecules and of water ranging from 7.5 to 45 and 55 to 92.5 %, respectively. However, the concentrations of macromolecules and water at the nanoscale within the various cell compartments are unknown. We recently developed a new approach, correlative cryo-analytical scanning transmission electron microscopy, for mapping the quantity of water within compartments previously shown to display GFP-tagged protein fluorescence on the same ultrathin cryosection. Using energy-dispersive X-ray spectrometry (EDXS), we then identified various elements (C, N, O, P, S, K, Cl, Mg) in these compartments and quantified them in mmol/l. Here, we used this new approach to quantify water and elements in the cytosol, mitochondria, condensed chromatin, nucleoplasm, and nucleolar components of control and stressed cancerous cells. The water content of the control cells was between 60 and 83 % (in the mitochondria and nucleolar fibrillar centers, respectively). Potassium was present at concentrations of 128-462 mmol/l in nucleolar fibrillar centers and condensed chromatin, respectively. The induction of nucleolar stress by treatment with a low dose of actinomycin-D to inhibit rRNA synthesis resulted in both an increase in water content and a decrease in the elements content in all cell compartments. We generated a nanoscale map of water and elements within the cell compartments, providing insight into their changes induced by nucleolar stress.

Targeted nano analysis of water and ions using cryocorrelative light and scanning transmission electron microscopy.

Cryo fluorescence imaging coupled with the cryo-EM technique (cryo-CLEM) avoids chemical fixation and embedding in plastic, and is the gold standard for correlated imaging in a close to native state. This multi-modal approach has not previously included elementary nano analysis or evaluation of water content. We developed a new approach allowing analysis of targeted in situ intracellular ions and water measurements at the nanoscale (EDXS and STEM dark field imaging) within domains identified by examination of specific GFP-tagged proteins. This method allows both water and ions- fundamental to cell biology- to be located and quantified at the subcellular level. We illustrate the potential of this approach by investigating changes in water and ion content in nuclear domains identified by GFP-tagged proteins in cells stressed by Actinomycin D treatment and controls. The resolution of our approach was sufficient to distinguish clumps of condensed chromatin from surrounding nucleoplasm by fluorescence imaging and to perform nano analysis in this targeted compartment.

Elastin peptides signaling relies on neuraminidase-1-dependent lactosylceramide generation.

The sialidase activity of neuraminidase-1 (Neu-1) is responsible for ERK 1/2 pathway activation following binding of elastin peptide on the elastin receptor complex. In this work, we demonstrate that the receptor and lipid rafts colocalize at the plasma membrane. We also show that the disruption of these microdomains as well as their depletion in glycolipids blocks the receptor signaling. Following elastin peptide treatment, the cellular GM(3) level decreases while lactosylceramide (LacCer) content increases consistently with a GM(3)/LacCer conversion. The use of lactose or Neu-1 siRNA blocks this process suggesting that the elastin receptor complex is responsible for this lipid conversion. Flow cytometry analysis confirms this elastin peptide-driven LacCer generation. Further, the use of a monoclonal anti-GM(3) blocking antibody shows that GM(3) is required for signaling. In conclusion, our data strongly suggest that Neu-1-dependent GM(3)/LacCer conversion is the key event leading to signaling by the elastin receptor complex. As a consequence, we propose that LacCer is an early messenger for this receptor.

Localization of Nopp140 within mammalian cells during interphase and mitosis.

We investigated distribution of the nucleolar phosphoprotein Nopp140 within mammalian cells, using immunofluorescence confocal microscopy and immunoelectron microscopy. During interphase, three-dimensional image reconstructions of confocal sections revealed that nucleolar labelling appeared as several tiny spheres organized in necklaces. Moreover, after an immunogold labelling procedure, gold particles were detected not only over the dense fibrillar component but also over the fibrillar centres of nucleoli in untreated and actinomycin D-treated cells. Labelling was also consistently present in Cajal bodies. After pulse-chase experiments with BrUTP, colocalization was more prominent after a 10- to 15-min chase than after a 5-min chase. During mitosis, confocal analysis indicated that Nopp140 organization was lost. The protein dispersed between and around the chromosomes in prophase. From prometaphase to telophase, it was also detected in numerous cytoplasmic nucleolus-derived foci. During telophase, it reappeared in the reforming nucleoli of daughter nuclei. This strongly suggests that Nopp140 could be a component implicated in the early steps of pre-rRNA processing.

Lumican core protein inhibits melanoma cell migration via alterations of focal adhesion complexes.

Lumican is a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM) with anti-tumor activity. We recently demonstrated that lumican inhibits the migration of melanoma cells and identified beta1 integrin as mediator of this effect [M.F. D'Onofrio, S. Brézillon, T. Baranek, C. Perreau, P.J. Roughley, F.X. Maquart, Y. Wegrowski, Identification of beta1 integrin as mediator of melanoma cell adhesion to lumican, Biochem. Biophys. Res. Commun. 365 (2008) 266-272]. The aim of the present work was to study beta1 integrin, focal adhesion complexes, actin distribution and expression in the presence of lumican substratum in comparison to type I collagen or fibronectin substrata in A375 human melanoma cells. The protein distribution was investigated by immunocytochemistry and confocal microscopy. In parallel, their expression was evaluated by Western immunoblotting and Real-time Reverse Transcription-PCR analyses. The interaction of melanoma cells with the lumican substratum resulted in heterogeneous distribution of beta1 integrin on cell membrane after 24h of seeding. Concomitantly, a reorganization of actin stress fibers and a significant decrease in vinculin immunostaining at focal adhesion complexes were observed. No alteration of the expression was detected at protein and mRNA levels. However, a cytosolic accumulation of vinculin focal adhesion protein was observed on lumican substratum by confocal microscopy. Moreover, vinculin expression was significantly increased in cytosolic fractions in comparison to cells seeded on type I collagen or fibronectin substrata. Our results suggest that lumican induces an alteration of the link between actin filaments and beta1 integrin, characterized by a cytosolic accumulation of vinculin focal adhesion protein, which could lead to a destabilization of focal adhesion complexes. In addition, focal adhesion kinase phosphorylated at tyrosine-397 (pFAK) was significantly decreased. Therefore, the cytoskeleton remodeling and the decreased pFAK phosphorylation induced by lumican in melanoma cells might explain, at least in part, the anti-invasive effect of this SLRP.

Tomography of the cell nucleus using confocal microscopy and medium voltage electron microscopy.

Changes in nuclear structures are widely used by pathologists as diagnostic and prognostic indicators in cancer cells. Recent studies have demonstrated that the cell nucleus is probably the most complex organelle in the cell. It contains the genome and is the site of all related activities such as DNA repair, DNA duplication, RNA synthesis, RNA processing and RNA transport. These activities take place within dynamic three-dimensional compartments. The detailed study of these compartments requires an approach termed "cell tomography" based on 3D imaging using confocal microscopy and electron tomography. In this paper, we will first summarize the most recent findings concerning the organization of the cell nucleus. We will then describe markers used to identify molecules specific for various nuclear compartments and their use in tomography of the cell nucleus by confocal microscopy and electron tomography.

Three-dimensional reconstruction of nucleolar components by electron microscope tomography.

The nucleus is a complex volume constituted of numerous subcompartments in which specific functions take place due to a specific spatial organization of their molecular components. To understand how these molecules are spatially organized within these machineries, it is necessary to investigate their three-dimensional organization at high resolution. To reach this goal, electron tomography appears to be a method of choice; it can generate tomograms with a resolution of a few nanometers by using multiple projections of a tilted section several hundred to several thousand nanometers in thickness imaged by transmission electron microscopy (TEM).Specific identification of molecules of interest contained within such thick sections requires their specific immunocytochemical labelling using electron-dense markers. We recently demonstrated that electron tomography of proteins immunostained with nanogold particles before embedding, and subsequently amplified with silver, was very fruitful due to the inherently high spatial resolution of the medium-voltage scanning and transmission electron microscope (STEM). Here we describe this approach, which is very efficient for tracing the 3D organization of proteins within complex machineries by using antibodies raised against one of the proteins, or against GFP to analyse GFP-tagged proteins.

Isolation of nucleoli from Ehrlich ascites tumor cells and dynamics of nascent RNA within isolated nucleoli.

Here we describe a new, rapid method for isolating nucleoli from Ehrlich tumor cells that preserves their morphological integrity and high transcriptional activity. Until now, methods for isolation of nucleoli were generally assumed to empty one of their three main compartments, the fibrillar center, of its contents. This new method consists of sonicating cells in an isotonic medium containing MgSO(4), spermidine, and spermine, followed by separation of nucleoli through a Percoll density gradient. Using the nonisotopic approach of labelling with BrUTP, we have further investigated the dynamics of nascent ribosomal RNAs (rRNAs) within morphologically intact isolated nucleoli at the electron microscope level. We show that ribosomal transcripts are elongated in the cortex of the fibrillar center and then enter the surrounding dense fibrillar component.

Deletion 2q36.2q36.3 with multiple renal cysts and severe mental retardation.

Interstitial 2q36 deletion is a rare event. We report on a patient with a de novo del(2)(q36.2q36.3) interstitial deletion of the long arm of chromosome 2 diagnosed by classical banding. The phenotype comprised facial dysmorphism, enlarged kidneys with multiple renal cysts, abnormal minora labia, asymmetric lower limbs with dysplastic patella, and severe mental retardation. By physical mapping, using array-comparative genomic hybridisation (CGH) confirmed by Fluorescent In Situ Hybridisation (FISH), the breakpoints of the deletion were mapped and the size of the deletions was measured: 5.61+/-0.19Mb. A skin biopsy was analysed using electronic microscopy showing an alteration of the structure and organisation of the dermal and peri-neuronal basement membrane. The relation between the phenotype and the deletion of both COL4A4 and COL4A3 genes, located in 2q36.3 loci, as well as the disruption of TRIP12 were discussed.

Prognostic value of a proliferation index including MIB1 and argyrophilic nucleolar organizer regions proteins in node-negative breast cancer.

OBJECTIVE: This study was designed to evaluate a cell proliferation marker, including the percentage of cycling cells (MIB1), and the duration of the cell cycle (assessed by argyrophilic nucleolar organizer regions proteins [AgNORs] measurement). STUDY DESIGN: We included 90 patients with invasive node-negative breast cancer. None received chemotherapy. With the help of a double-staining technique, a proliferation index (PI) was determined by multiplying the percentage of MIB1-positive cells by the mean area of the AgNORs present in those MIB1-positive cells. PI was evaluated for its impact on overall survival (OS) and disease-free survival (DFS). RESULTS: We demonstrated that PI was correlated to OS. For DFS, it conserved its high prognostic value only in univariate analysis. The global amount of AgNORs was more discriminative for DFS. CONCLUSION: PI and AgNOR quantification supplied additional prognosis information in node-negative patients, and we propose to integrate them in further studies.

A protocol for studying the kinetics of RNA within cultured cells: application to ribosomal RNA.

This protocol describes a nonisotopic method for high-resolution investigation of the kinetics of RNA within the cell. This involves the incorporation of bromouridine-5'-triphosphate into RNA of living cells by lipofection followed by immunocytological detection of BrRNAs. The use of the same antibody identified either with fluorescence or with gold particles revealed the three-dimensional organization of sites containing labeled RNAs or their precise localization by using confocal and ultrastructural microscopy, respectively. Comparison of three-dimensional reconstruction obtained from the series of optical sections and ultrathin sections was extremely fruitful to describe topological and spatial dynamics of RNAs from their synthesis site inside the nucleus to the cytoplasm. Combined with immunolocalization of proteins involved in different nuclear activities and with highly resolved three-dimensional visualizations of the labelings, this method should also provide a significant contribution to our understanding of the functional, volumic organization of the cell nucleus. The entire protocol can be completed in approximately 10 d.

Pleomorphic hyalinizing angiectatic tumor of soft parts: ultrastructural analysis of a case with original features.

Pleomorphic hyalinizing angiectatic tumor, a rare neoplasm of uncertain lineage resembling malignant fibrous histiocytoma and schwannoma, was first described in 1996 by M. E. F. Smith et al. (Am Surg Pathol. 20:21-29). To date, less than 100 cases have been reported in the international literature. It occurs in subcutaneous and intramuscular soft tissues of extremities or trunk in adults without sex predilection. All lesions are composed of sheets and fascicles of spindled and pleomorphic cells associated with clusters of thick-walled ectatic vessels surrounded by a perivascular hyaline material and inflammatory cells such as mast cells. About one-half of these neoplasms express CD34. No patient has developed metastases but occasional local recurrences are possible. This tumor of uncertain lineage is suggested to be an aggressive locally growing low-grade sarcoma. Only 3 cases were previously studied by electron microscopy and appeared to consist of primitive fibroblastic cells. The authors report histological and ultrastructural characteristics of a new case of PHAT excised from the right buttock of a 66-year-old man with the presence of ganglion-like cells, a feature that has not been previously reported, and unusual central ischemic necrosis. The features of this case are suggestive of a fibroblastic origin.