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Summary: The prognosis of patients with glioblastoma (GBM) remains dismal, with a median survival of approximately 15 months. Current preclinical GBM models are limited by the lack of a “normal” human microenvironment and the inability of many tumor cell lines to accurately reproduce GBM biology. To address these limitations, we have established a model system whereby we can retro-engineer patient-specific GBMs using patient-derived glioma stem cells (GSCs) and human embryonic stem cell (hESC)-derived cerebral organoids. Our cerebral organoid glioma (GLICO) model shows that GSCs home toward the human cerebral organoid and deeply invade and proliferate within the host tissue, forming tumors that closely phenocopy patient GBMs. Furthermore, cerebral organoid tumors form rapidly and are supported by an interconnected network of tumor microtubes that aids in the invasion of normal host tissue. Our GLICO model provides a system for modeling primary human GBM ex vivo and for high-throughput drug screening. : To address limitations with current preclinical glioblastoma (GBM) models, Linkous et al. establish a “GLICO” (cerebral organoid glioma) model to retro-engineer patient-specific GBMs using patient-derived glioma stem cells and human cerebral organoids. Resulting tumors closely phenocopy patient GBMs and are supported by tumor microtubes that promote invasion into host tissue. Keywords: cerebral organoids, glioma stem cells, glioblastoma, glioma, tumor microtubes, human embryonic stem cells, brain tumors, stem-cell-based disease models, tissue engineering, cancer stem cells

Background: Obesity is associated with increased breast cancer incidence and progression. Breast adipose tissue produces a number of factors hypothesized to contribute to this observation, including estrogens, adipokines, inflammatory mediators, and free fatty acids. Adipose tissue also releases extracellular vesicles (EVs) that can act locally or circulate to distant sites. EVs are nano-sized particles that are characterized by their lipid bilayer and contain nucleic acids, proteins, lipids and other molecules that can affect target cells. Recently, EVs derived from adipose tissue have received increasing attention and numerous studies have been conducted to explore the relationship between adipose tissue-derived EVs and different types of cancers, including breast cancer. Here, we provide novel insights into the sustained effects of EVs, collected from fresh breast adipose tissue, via effects on mitochondrial mass and respiration in estrogen receptor (ER)-positive breast cancer cells. Methods: EVs were collected from fresh breast adipose tissue from reduction mammoplasties. Long-term education was performed by treating MCF7 and T47D breast cancer cell lines with 3 doses of EVs over the course of 7 days. xCelligence was used to quantify cell proliferation. RNA Seq was performed on one of the educated MCF7 pairs. Mitochondrial respiration was evaluated using the Seahorse XF instrument. MitoTracker Green fluorescent staining and transmission electron microscopy (TEM) were used to assess mitochondrial density and morphology. Western blotting was used to identify pathways that may be involved in the effects of adipose tissue derived EVs on breast cancer cells. Results: EVs derived from adipose tissue of overweight/obese individuals (O-EVs) stimulate proliferation of MCF7 breast cancer cells compared with that from lean individuals (L-EVs). Compared with controls, O-EVs also induce proliferation of T47D breast cancer cells. RNA-Seq data reveal that genes involved in oxidative phosphorylation (OXPHOS) are significantly upregulated in O-EV-treated MCF7 cells compared to control. Compared with control cells, basal mitochondrial respiration of O-EV-treated MCF7 cells is significantly higher than control. Metformin, which inhibits mitochondrial complex I and ATP synthase, inhibits the O-EV-stimulated proliferation of MCF7 cells, while having no effect on the proliferation of control cells. Both MitoTracker Green fluorescent staining and TEM demonstrate increased mitochondrial mass/number. Western blotting reveals that O-EVs significantly increase phosphorylation of Akt, which is the major upstream regulator of mTOR signaling, and phosphorylation of P70 S6 kinase and 4EBP1, which are two main downstream effectors of mTOR signaling, affecting both protein synthesis and mitochondrial respiration. Conclusions: Breast adipose tissue EVs from overweight and obese women stimulate the proliferation of ER+ breast cancer cells by increasing mitochondrial mass and stimulating mitochondrial respiration, providing a novel mechanistic link between obesity and breast cancer. Our studies also suggest that metformin or mTOR-targeting drugs, may prove useful to break obesity-breast cancer link. Support: NIH R01CA215797, Anne Moore Breast Cancer Research Fund Citation Format: Shuchen Liu, Alberto Benito-Martin, Phoebe Piloco, Catherine Liu, Paul Paik, Jason A Spector, Fanny A Pelissier Vatter, David Lyden, David M Otterburn, Leslie Cohen, Olivier Elemento, Rohan Bareja, Leona Cohen-Gould, Samuel Calto, Kristy A Brown. Extracellular vesicles from obese human breast adipose tissue promote breast cancer cell proliferation by increasing mitochondrial mass and stimulating mitochondrial respiration [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-05-02.

Granular cell tumors (GCTs) are rare tumors that can arise in multiple anatomical locations, and are characterized by abundant intracytoplasmic granules. The genetic drivers of GCTs are currently unknown. Here, we apply whole-exome sequencing and targeted sequencing analysis to reveal mutually exclusive, clonal, inactivating somatic mutations in the endosomal pH regulators ATP6AP1 or ATP6AP2 in 72% of GCTs. Silencing of these genes in vitro results in impaired vesicle acidification, redistribution of endosomal compartments, and accumulation of intracytoplasmic granules, recapitulating the cardinal phenotypic characteristics of GCTs and providing a novel genotypic–phenotypic correlation. In addition, depletion of ATP6AP1 or ATP6AP2 results in the acquisition of oncogenic properties. Our results demonstrate that inactivating mutations of ATP6AP1 and ATP6AP2 are likely oncogenic drivers of GCTs and underpin the genesis of the intracytoplasmic granules that characterize them, providing a genetic link between endosomal pH regulation and tumorigenesis., Granular cell tumors (GCTs) are rare tumors that arise in multiple anatomical locations. Here, the authors investigate the genomics of GCTs, finding inactivating somatic mutations in ATP6AP1 or ATP6AP2 in 72% of the 82 GCTs analyzed. In vitro manipulation of these genes recapitulated GCT phenotypes in cellular models.

Small heat shock proteins are chaperones with variable mechanisms of action. The function of cardiac family member Hspb7 is unknown, despite being identified through GWAS as a potential cardiomyopathy risk gene. We discovered that zebrafish hspb7 mutants display mild focal cardiac fibrosis and sarcomeric abnormalities. Significant mortality was observed in adult hspb7 mutants subjected to exercise stress, demonstrating a genetic and environmental interaction that determines disease outcome. We identified large sarcomeric proteins FilaminC and Titin as Hspb7 binding partners in cardiac cells. Damaged FilaminC undergoes autophagic processing to maintain sarcomeric homeostasis. Loss of Hspb7 in zebrafish or human cardiomyocytes stimulated autophagic pathways and expression of the sister gene encoding Hspb5. Inhibiting autophagy caused FilaminC aggregation in HSPB7 mutant human cardiomyocytes and developmental cardiomyopathy in hspb7 mutant zebrafish embryos. These studies highlight the importance of damage-processing networks in cardiomyocytes, and a previously unrecognized role in this context for Hspb7.

The innate immune system has been implicated in both AKI and CKD. Damaged mitochondria release danger molecules, such as reactive oxygen species, DNA, and cardiolipin, which can cause NLRP3 inflammasome activation and upregulation of IL-18 and IL-1β. It is not known if mitochondrial damage persists long after ischemia to sustain chronic inflammasome activation. We conducted a 9-month study in Sprague–Dawley rats after 45 minutes of bilateral renal ischemia. We detected glomerular and peritubular capillary rarefaction, macrophage infiltration, and fibrosis at 1 month. Transmission electron microscopy revealed mitochondrial degeneration, mitophagy, and deformed foot processes in podocytes. These changes progressed over the study period, with a persistent increase in renal cortical expression of IL-18, IL-1β, and TGF-β, despite a gradual decline in TNF-α expression and macrophage infiltration. Treatment with a mitoprotective agent (SS-31; elamipretide) for 6 weeks, starting 1 month after ischemia, preserved mitochondrial integrity, ameliorated expression levels of all inflammatory markers, restored glomerular capillaries and podocyte structure, and arrested glomerulosclerosis and interstitial fibrosis. Further, helium ion microscopy vividly demonstrated the restoration of podocyte structure by SS-31. The protection by SS-31 was sustained for ≥6 months after treatment ended, with normalization of IL-18 and IL-1β expression. These results support a role for mitochondrial damage in inflammasome activation and CKD and suggest mitochondrial protection as a novel therapeutic approach that can arrest the progression of CKD. Notably, SS-31 is effective when given long after AKI and provides persistent protection after termination of drug treatment.

Following murine injection, the ultrastructural loci of nanoparticles (NPs) containing lutetium-177 (Lu-177) conjugated to an anti-thrombomodulin antibody (mAb-201b) were determined. The results confirmed prior work localizing NPs using Single Photon Emission Computed Tomography (SPECT) scans. The in vivo pharmacokinetics of these NPs were also identified. mAb-201b antibodies are primarily attracted to the thrombomodulin, a membrane protein in the endothelium of the lung vasculature. SPECT images demonstrated NPs in the lungs, liver, spleen, and proximal small bowel. Prior injection of clodronate liposomes reduced the number of circulating macrophages, which, in turn, reduced NP phagocytosis. At 24 h after injection of NPs and after final SPECT imaging, the lungs, liver, spleen, and kidneys were harvested for transmission electron microscopy. Although some NPs were found in all four organs, 85% of the injected dose was localized in type I and type II pneumocytes. Small concentrations were found in secondary lysosomes in hepatocytes, in splenic macrophages, and in an intravascular macrophage in a kidney. Importantly, there was no apoptosis or necrosis in any of the tissues, highlighting the relative safety of the radionuclide NP, whose primary interaction with non-targeted organs/tissues is in the filtration process. In addition to validating the biodistribution results of the SPECT scans carried out in our prior work, this study is proof of principle that NPs conjugated with appropriate antibodies can target specific antigens in vivo. From a theranostic perspective, these results suggest that radioactive nanoconjugates labeled with proper antigens should be able to target and destroy a variety of cancers with minimal harm to the surrounding healthy cells.

In our Core Facility, we are presented with a wide variety of tissue and sample types to process for TEM observation. Some tissues, such as liver, do not present orientation problems. Other tissues such as skeletal muscle have specific orientations that must be maintained. As long as the initial dissection of the tissue is done so that the orientation or area of interest can be identified, these samples do not present a problem. Likewise, when it is not necessary to maintain orientation, cells in culture can be treated with trypsin, to release them from the plate, and pelleted. Or the monolayer can be fixed in vitro and then scraped and pelleted, and the pellet can be processed while adherent to the wall of the Eppendorf tube or handled like a small piece of tissue, among other methods.

The heterogeneity of exosomal populations has hindered our understanding of their biogenesis, molecular composition, biodistribution and functions. By employing asymmetric flow field-flow fractionation (AF4), we identified two exosome subpopulations (large exosome vesicles, Exo-L, 90–120 nm; small exosome vesicles, Exo-S, 60–80 nm) and discovered an abundant population of non-membranous nanoparticles termed ‘exomeres’ (~35 nm). Exomere proteomic profiling revealed an enrichment in metabolic enzymes and hypoxia, microtubule and coagulation proteins as well as specific pathways, such as glycolysis and mTOR signalling. Exo-S and Exo-L contained proteins involved in endosomal function and secretion pathways, and mitotic spindle and IL-2/STAT5 signalling pathways, respectively. Exo-S, Exo-L and exomeres each had unique N-glycosylation, protein, lipid, DNA and RNA profiles and biophysical properties. These three nanoparticle subsets demonstrated diverse organ biodistribution patterns, suggesting distinct biological functions. This study demonstrates that AF4 can serve as an improved analytical tool for isolating extracellular vesicles and addressing the complexities of heterogeneous nanoparticle subpopulations.

Refined cancer models are needed to bridge the gaps between cell line, animal and clinical research. Here we describe the engineering of an organotypic colon cancer model by recellularization of a native human matrix that contains cell-populated mucosa and an intact muscularis mucosa layer. This ex vivo system recapitulates the pathophysiological progression from APC-mutant neoplasia to submucosal invasive tumor. We used it to perform a Sleeping Beauty transposon mutagenesis screen to identify genes that cooperate with mutant APC in driving invasive neoplasia. We identified 38 candidate invasion-driver genes, 17 of which, including TCF7L2, TWIST2, MSH2, DCC, EPHB1 and EPHB2 have been previously implicated in colorectal cancer progression. Six invasion-driver genes that have not, to our knowledge, been previously described were validated in vitro using cell proliferation, migration and invasion assays and ex vivo using recellularized human colon. These results demonstrate the utility of our organoid model for studying cancer biology.

proNGF and p75NTR are induced following fatal myocardial infraction and are required for the development of microvascular injury., Treatment of acute cardiac ischemia focuses on reestablishment of blood flow in coronary arteries. However, impaired microvascular perfusion damages peri-infarct tissue, despite arterial patency. Identification of cytokines that induce microvascular dysfunction would provide new targets to limit microvascular damage. Pro–nerve growth factor (NGF), the precursor of NGF, is a well characterized cytokine in the brain induced by injury. ProNGF activates p75 neurotrophin receptor (p75NTR) and sortilin receptors to mediate proapoptotic responses. We describe induction of proNGF by cardiomyocytes, and p75NTR in human arterioles after fatal myocardial infarction, but not with unrelated pathologies. After mouse cardiac ischemia-reperfusion (I-R) injury, rapid up-regulation of proNGF by cardiomyocytes and p75NTR by microvascular pericytes is observed. To identify proNGF actions, we generated a mouse expressing a mutant Ngf allele with impaired processing of proNGF to mature NGF. The proNGF-expressing mouse exhibits cardiac microvascular endothelial activation, a decrease in pericyte process length, and increased vascular permeability, leading to lethal cardiomyopathy in adulthood. Deletion of p75NTR in proNGF-expressing mice rescues the phenotype, confirming the importance of p75NTR-expressing pericytes in the development of microvascular injury. Furthermore, deficiency in p75NTR limits infarct size after I-R. These studies identify novel, nonneuronal actions for proNGF and suggest that proNGF represents a new target to limit microvascular dysfunction.

Contact-dependent communication between immune cells generates protection but also facilitates viral spread. Here we found that macrophages formed long-range actin-propelled conduits in response to negative factor (Nef), a human immunodeficiency virus type 1 (HIV-1) protein with immunosuppressive functions. Conduits attenuated immunoglobulin G2 (IgG2) and IgA class switching in systemic and intestinal lymphoid follicles by shuttling Nef from infected macrophages to B cells through a guanine-exchange factor-dependent pathway involving the amino-terminal anchor, central core and carboxy-terminal flexible loop of Nef. By showing stronger virus-specific IgG2 and IgA responses in patients with Nef-deficient virions, our data suggest that HIV-1 exploits intercellular 'highways' as a 'Trojan horse' to deliver Nef to B cells and evade humoral immunity systemically and at mucosal sites of entry.

The immunophilin-ligand FK506 has been shown to ameliorate erectile function and preserve cavernous nerve (CN) architecture in short-term-studies using rat models of CN injury.The aim of this series was to ascertain the optimal dose and timing of FK506 administration in this animal model.Rats underwent bilateral CN crush and were treated with FK506 at different time points. There were control (C) and sham groups for each time point. Based on preliminary experiments, the CN-crush rats had no treatment (C) or either FK506 1 mg/kg (BL) or 3.2 mg/kg (BH) for 3 days prior to and the day of CN crush (PRE), on the day of and for 3 days following CN crush (POST) and for 3 days pre-, on the day of, and 3 days post-CN crush (PP).All animals had measurement of intracavernosal pressure/mean arterial blood pressure (ICP/MAP) ratios at 28 days post-CN crush. Structural analysis was conducted in the POST groups. Penile tissue was assessed for apoptosis with terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay and immunohistochemically for neural factors (growth associated protein 43 [GAP43], nerve growth factor [NGF], and neural nitric oxide synthase [nNOS]). The CN architecture was examined by transmission electron microscopy (TEM).Sham animals had an ICP/MAP ratio of 70%. Only the BH-POST group revealed an improved ICP/MAP ratio compared with C (50 +/- 9% vs. 32 +/- 8%, P0.01). nNOS staining was significantly restored reaching sham levels in BL-POST and BH-POST groups vs. C (P0.05). NGF and GAP43 staining displayed no significant differences between C and treatment groups (P0.05). Apoptosis was significantly reduced in BL-POST and BH-POST groups compared with C (16 +/- 4%, 21 +/- 9%, and 63 +/- 7%, P0.001). TEM exhibited preservation of CN architecture for BH-POST compared with C.These results suggest that short-term treatment with doses of FK506 higher than previously utilized preserves erectile function in the rat CN-injury model. Pretreatment appears to offer no advantage. However, FK506 administration just prior to CN injury and for a short-time post-injury achieves the best functional and structural preservation outcomes.

Introduction Radical prostatectomy (RP) is associated with erectile dysfunction (ED). A single, placebo-controlled, human study has assessed the effects of regular sildenafil use after RP and demonstrated an increased chance of preservation of preoperative erectile function. Aim This study was undertaken to define the effects of such a regimen in an animal model. Methods Using the cavernous nerve (CN) crush injury model, animals were divided into a number of groups: no CN injury (sham), bilateral CN injury exposed to either no sildenafil (control) or sildenafil at two doses (10 and 20 mg/kg) subcutaneously daily for three different durations (3, 10, 28 days). Main Outcome Measures At these time points, CN electrical stimulation was used to assess erectile function by mean intracavernosal pressure (ICP)/mean arterial pressure (MAP) ratio. For the structural analyses, whole rat penes were harvested. Staining for Masson's trichrome was utilized to calculate the smooth muscle-collagen ratio. Immunohistochemical antibody staining was performed for endothelial (CD31 and eNOS) and neural (GAP43, NGF, and nNOS) factors and immunoblotting was performed to analyze the AKT/eNOS pathway. Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) assay was used for the assessment of apoptotic indices and the CN architecture was evaluated by transmission electron microscopy (TEM). Results Erectile function was improved with sildenafil in a time- and dose-dependent fashion with maximization of erectile function recovery occurring with daily 20 mg/kg at the 28-day time point. Sildenafil use resulted in smooth muscle-collagen ratio protection and CD31 and eNOS expression preservation. Sildenafil reduced apoptotic indices significantly compared with control. Animals exposed to sildenafil had increased phosphorylation of akt and eNOS. Tem demonstrated distinct differences in architecture between control and sildenafil groups toward an increased amount of myelinized nerve fibers. Conclusions Sildenafil use in the CN crush injury model preserves erectile function that appears to be mediated predominantly through preservation of smooth muscle content and endothelial function as well as through reduction in apoptosis. Mulhall JP, Muller A, Donohue JF, Mullerad M, Kobylarz K, Paduch DA, Tal R, Li PS, Cohen-Gould L, and Scardino PT. The functional and structural consequences of cavernous nerve injury are ameliorated by sildenafil citrate.

Bovine Viral Diarrhea Virus (BVDV) is a positive sense, single-stranded RNA virus which exhibits two biotypes in standard cell culture systems. The cytopathic strains of this virus (cpBVDV) induce dramatic cytoplasmic vacuolization in cell cultures, while infection with the non-cytopathic (NCP-BVDV) strains produces no overt changes in the host cells. Our results show that extensive cytoplasmic vacuolization is the earliest morphological change in response to cpBVDV infection in MDBK cells. Cells with extensive vacuolization showed no co-existing chromatin condensation, caspase activation, or loss of membrane integrity. In addition, the caspase inhibitor (zVAD-fmk), although improving cell viability of infected cells from 6.7+/-2.2% to 18.8+/-2.2%, did not prevent vacuolization. On the ultrastructural level, the virus-induced cytoplasmic vacuoles are single membrane structures containing organelles and cellular debris, which appear capable of fusing with other vacuoles and engulfing surrounding cytoplasmic materials. LysoTracker Red which marks lysosomes did not stain the virus-induced cytoplasmic vacuoles. In addition, this lysosomal dye could be observed in the cytoplasm of vacuolized cells, suggesting a lysosomal abnormality. Our data demonstrate that cpBVDV induced a novel cell death pathway in MDBK cells that is primarily associated with lysosomal dysfunction and the formation of phagocytic cytoplasmic vacuoles, and this mode of cell death is different from apoptosis and necrosis.

Nestin, a marker of multi-lineage stem and progenitor cells, is a member of intermediate filament family, which is expressed in neuroepithelial stem cells, several embryonic cell types, including mesonephric mesenchyme, endothelial cells of developing blood vessels, and in the adult kidney. We used Nestin-green fluorescent protein (GFP) transgenic mice to characterize its expression in normal and post-ischemic kidneys. Nestin-GFP-expressing cells were detected in large clusters within the papilla, along the vasa rectae, and, less prominently, in the glomeruli and juxta-glomerular arterioles. In mice subjected to 30 min bilateral renal ischemia, glomerular, endothelial, and perivascular cells showed increased Nestin expression. In the post-ischemic period, there was an increase in fluorescence intensity with no significant changes in the total number of Nestin-GFP-expressing cells. Time-lapse fluorescence microscopy performed before and after ischemia ruled out the possibility of engraftment by the circulating Nestin-expressing cells, at least within the first 3 h post-ischemia. Incubation of non-perfused kidney sections resulted in a medullary-to-cortical migration of Nestin-GFP-positive cells with the rate of expansion of their front averaging 40 microm/30 min during the first 3 h and was detectable already after 30 min of incubation. Explant matrigel cultures of the kidney and aorta exhibited sprouting angiogenesis with cells co-expressing Nestin and endothelial marker, Tie-2. In conclusion, several lines of circumstantial evidence identify a sub-population of Nestin-expressing cells with the mural cells, which are recruited in the post-ischemic period to migrate from the medulla toward the renal cortex. These migrating Nestin-positive cells may be involved in the process of post-ischemic tissue regeneration.

The acronym for “certified electron microscopy technologist” is CEMT, and these letters designate the bearer as having passed the written and practical exams necessary to become “certified”! The acronym was approved by the MSA Council to more firmly provide a professional identification of certification in the area of transmission electron microscopy (TEM) of biological tissue. A CEMT may use these initials after his or her name for as long as the certification remains valid (for example, Jane Doe, AA, BS, CEMT).

Despite growing evidence for a mitochondrial localization of nitric oxide (NO) synthase and a broadening spectrum of NO actions on mitochondrial respiration and apoptosis, the basis for interaction between the enzyme and the organelle remain obscure. Here we investigated mitochondrial localization of endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells and human embryonic kidney cells transfected or infected with eNOS expression vectors. Copurification of eNOS with mitochondria was observed in both human umbilical vein endothelial cells and eNOS-expressing human embryonic kidney cells. Immunodetectable eNOS was cleaved from mitochondria by proteinase K treatment, suggesting eNOS association with the outer mitochondrial membrane. Localization of eNOS to a proteinase K-cleavable site on the cytoplasmic face of the outer membrane was confirmed by immunogold labeling of non-permeabilized mitochondria. Markers for mitochondrial subfractions ruled out the possibility of eNOS association with an intramitochondrial site or inverted mitochondrial particles. Denaturation of eNOS did not attenuate association with mitochondria. Mutant eNOS lacking a pentabasic amino acid sequence within the autoinhibitory domain (residues 628-632 of the bovine eNOS) showed dramatically reduced binding to the mitochondrial but not to the plasma membrane, which was associated with increased oxygen consumption. Collectively, these findings argue in favor of eNOS localization to the outer mitochondrial membrane in endothelial cells and identify elements of a novel anchoring mechanism.

Stromal-epithelial interactions, which regulate the migration of prostate epithelial cells, play an important role in prostate development, prostatic hyperplasia, and prostate cancer. The objective of this study was to determine how the prostate stroma stimulates the migration of primary prostate epithelial cells (PECs). In the Boyden chamber assay, PEC migration was strongly induced by the conditioned medium of primary prostate stromal cells (PSC-CM). Stimulation of PEC migration depended on the concerted action of adhesion and motility factors in the PSC-CM. Immobilized proteins from PSC-CM mediated adhesion, spreading, and head-to-tail polarization of PECs. Migration induced by immobilized PSC-CM proteins was significantly increased by hepatocyte growth factor/scatter factor (HGF/SF). Inhibition of P13-kinase or Src-family kinases, but not MEK or PLCchi, abolished migration in the Boyden chamber assay. Consistent with their concerted activity in migration assays, the combination of adhesion and motility factors was required for efficient activation of the P13-kinase/Akt pathway. HGF/SF in the PSC-CM was the principal stimulator of the P13-kinase/Akt pathway and an important mediator of PSC-CM-induced PEC migration. In conclusion, our data show that the migration of primary PECs is regulated by the P13-kinase and Src-family kinase signaling pathways and that the activation of the P13-kinase pathway requires adhesion and motility factors from the prostate stroma.

Department of Cell Biology and Anatomy, Weill MedicalCollege of Cornell University New York, NY, USA.SummaryThe ability to gain entry and resist the antimicrobialintracellular environment of mammalian cells is anessential virulence property of Mycobacterium tuber-culosis. A purified recombinant protein expressed bya 1362 bp locus (mce1) in the M. tuberculosis genomepromoted uptake into HeLa cells of polystyrene latexmicrospheres coated with the protein. N-terminusdeletion constructs of Mce1 identified a domainlocated between amino acid positions 106 and 163that was needed for this cell uptake activity. Mce1contained hydrophobic stretches at the N-terminuspredictive of a signal sequence, and colloidal goldimmunoelectron microscopy indicated that the corre-sponding native protein is expressed on the surfaceof the M. tuberculosis organism. The complete M.tuberculosis genome sequence revealed that it con-tained four homologues of mce (mce1, mce2, mce3,mce4) and that they were all located within operonscomposed of genes arranged similarly at differentlocations in the chromosome. Recombinant Mce2,which had the highest level of identity (67%) to Mce1,was unable to promote the association of microsphereswith HeLa cells. Although the exact function of Mce1 isstill unknown, it appears to serve as an effector moleculeexpressed on the surface of M. tuberculosis that iscapable of eliciting plasma membrane perturbationsin non-phagocytic mammalian cells.IntroductionAlthough it is generally accepted that the primary cell typeencountered by Mycobacterium tuberculosis during infec-tion in vivo is the alveolar macrophage, this organism canenter a variety of cell types in vitro. In the 1950s, Shepard(1957; 1958) showed that M. tuberculosis can entermonolayers of HeLa, monkey kidney and human amnioncells and, more recently, McDonough and Kress (1995)showed that the organism enters lung epithelial cells.None of these cell types is considered to be truly phagocyticand, therefore, the entry of M. tuberculosis into such cellsmust be mediated by the organism itself. Although it is notclear whether the ability of M. tuberculosis to enter non-phagocytic cells has any physiological or pathologicalsignificance, Shepard (1957) suggested that the pathogeni-city of mycobacteria for humans is generally correlated withthe ability of the organism to multiply inside HeLa cells. Thein vitro observation made with non-phagocytic cells maytherefore be taken advantage of as a way to search forM. tuberculosis products that trigger mammalian cells totake up the bacillus. Such products may mediate bacterialuptake by eliciting cell signalling events that resemblethose stimulated by invasive enteric bacterial pathogensin their interaction with non-phagocytic mammalian cells(Bliska et al., 1993; Rosqvist et al., 1994; Persson et al.,1995; Galan, 1996; Hakansson et al., 1996; Menard et al.,1996). Once identified, such products, or bacterial effectormolecules, may be analysed further for their effect onmacrophages, the natural target of M. tuberculosis.A DNA fragment of a strain of M. tuberculosis clonedinto a non-pathogenic Escherichia coli strain was reportedpreviously to confer upon the E. coli host an ability to enterHeLa cells (Arruda et al., 1993). The genetic locusassociated with HeLa cell invasion was confined to anupstream locus in a 1535 bp SauIIIa fragment. In thisreport, we describe a 1362 bp open reading frame (ORF)that we call the mycobacterium cell entry (mce) gene,which encodes a protein capable of facilitating the uptakeof latex microspheres into non-phagocytic cells. This M.tuberculosis protein was found to be expressed by a genebelonging to a family of genes that may encode proteinslocalized to the surface of the organism (Cole et al., 1998).ResultsExpression and purification of the recombinant Mce1protein (rMce1)An

For years, I have used a hybrid Epon-analog resin to embed in culture dishes. I use a standard Epon formula but utilize the following components: LX-112 and DMP-30 from Ladd Research Industries DDSA and NMA from Electron Microscopy Sciences I know it seems weird, but years ago I tried all sorts things, from the “straight” formulations from each vendor to a bunch of mixtures. This one has never reacted with the plastic of the dishes. Here is how I do the actual embedding of the cell monolayers in the dishes: 1) After the last 100% ethanol, remove the alcohol and cover the bottom of the well with a layer of resin mixture that is about 2 mm deep. 2) Insert embedding tubes that are made by cutting the pyramidal bottoms off of BEEM capsules (just slice them with a fresh razor blade and be sure to insert them so that the manufactured end rather than the cut one is sitting against the dish). 3) After inserting labels into the tubes, put them into the oven at 60° overnight. 4) In the morning, fill just the embedding tubes and return everything to the oven again to finish polymerizing. 5) When the resin is cured, grab the tubes with a pair of needlenosed pliers and snap them out. Sometimes a bit of the bottom of the dish comes away with the block, but often a very smooth block face results. If some of the dish comes up, it is easy to see under a dissecting microscope, and the dish portion comes away easily when trimming the block face. I often cut away part of the block face with a jeweler's saw, either to keep it in reserve or to re-embed it in order to get cross sections, and then trim the rest into a narrow rectangle.

The retinal pigment epithelium is endowed with a unique distribution of certain plasma membrane proteins. Na+,K+-ATPase, for instance, is polarized to the apical surface of RPE, rather than to the basolateral surface as in most other epithelia. To study the sorting pathways of RPE cells, we used temperature sensitive mutants of influenza and vesicular stomatitis virus (VSV) to synchronize the transport of hemagglutinin (HA) and VSV G protein (VSV G) along the biosynthetic pathway of the RPE cell line RPE-J. After HA and VSV G accumulated in the trans-Golgi network of RPE-J cells kept at 20 degrees C, transfer to the permissive temperature (32 degrees C) resulted in the transport of both HA and VSV G to the basolateral plasma membrane. Later, while VSV G remained basolateral, HA progressively reversed its polarity, eventually becoming apical. Further analysis demonstrated that the reversal of HA polarity was due to transcytosis of HA from the basolateral to the apical surface of RPE-J cells. To determine whether HA followed a transcytotic route in RPE in vivo, influenza and VSV were injected into the subretinal space of rat eyes. Again, both HA and VSV G were initially observed at the basolateral surface of RPE cells. However, whereas VSV G remained there, HA progressively redistributed to the apical surface. These findings demonstrated that RPE cells use a transcytotic pathway for the targeting of at least some apical proteins to their destination.

Abstract Heart contraction is coordinated by conduction of electrical excitation through specialized tissues of the cardiac conduction system. By retroviral single-cell tagging and lineage analyses in the embryonic chicken heart, we have recently demonstrated that a subset of cardiac muscle cells terminally differentiates as cells of the peripheral conduction system (Purkinje fibers) and that this occurs invariably in perivascular regions of developing coronary arteries. Cis regulatory elements that function in transcriptional regulation of cells in the conducting system have been distinguished from those in contractile cardiac muscle cells; eg, 5′ regulatory sequences of the desmin gene act as enhancer elements in skeletal muscle and in the conduction system but not in cardiac muscle. We hypothesize that Purkinje fiber differentiation involves a switch of the gene expression program from that characteristic of cardiac muscle to one typical of skeletal muscle. To test this hypothesis, we examined the expression of myosin binding protein-H (MyBP-H) in Purkinje fibers of chicken hearts. This unique myosin binding protein is present in skeletal but not cardiac myocytes. A site-directed polyclonal antibody (AB105) was generated against MyBP-H. Immunohistological analysis of the myocardium mapped the AB105 antigen predominantly to A bands of myofibrils within Purkinje fibers. Western blot analysis of whole extracts from the ventricular wall of adult chicken hearts revealed that the AB105 epitope was restricted to a single protein of ≈86 kD, the same size as MyBP-H in skeletal muscle. Biochemical properties of the Purkinje fiber 86-kD protein and RNase protection analyses of its mRNA indicate that Purkinje fiber 86-kD protein is indistinguishable from skeletal muscle MyBP-H. The results provide evidence that skeletal muscle MyBP-H is expressed in a subset of cardiac muscle cells that differentiate into Purkinje fibers of the heart.

In gastrulation stage embryos of birds and mammals, the heart field is established as mesodermal crescents flanking the area rostrolateral to Hensen's node. Subsequent fusion of the bilateral heart primordia gives rise to a single tubular heart consisting of two epithelial layers: an outer myocardium and an inner endocardium. To date, it is uncertain whether these two distinct cell types of the heart arise from common or separate progenitor populations of mesodermal cells within the heart field. By retroviral single cell marking and tracking, we examined the diversity of cell populations present in the heart field of stage 4 chicken embryos. Here we demonstrate that individual mesodermal cells in the heart field gave rise to a clone consisting only of one cell type, either endocardial or myocardial cells; i.e., 95.1% of the mesoderm-derived clones were localized in the myocardium, while 4.9% of them were found in endocardium. No clones containing both of these two cell types were detected. The results suggest that the heart field mesoderm at stage 4 consists of at least two distinct subpopulations, containing more premyocardial cells than preendocardial cells. If there exists a common precursor of both myocardial and endocardial cells, the lineage diversification must occur at or prior to the arrival of mesodermal cells to the heart field.

Rat retinal pigment epithelial (RPE) cells were immortalized by infection with a temperature-sensitive tsA SV40 virus and following cloning and selection for epithelial properties the polarized RPE-J cell line was obtained. At the permissive temperature of 33 degrees C, RPE-J cells behave as an immortalized cell line. When RPE-J cells are grown on nitrocellulose filters coated with a thin layer of Matrigel in the presence of 10(−8) M retinoic acid for 6 days at 33 degrees C and then switched for 33–36 hours to the non-permissive temperature of 40 degrees C, they acquire a differentiated polarized RPE phenotype. Under these growth conditions, RPE-J cells exhibit circumferential staining for the tight-junction protein ZO-1 and acquire a transepithelial resistance of 350 ohms cm2. Morphologically, RPE-J cells exhibit a characteristic RPE morphology with extensive apical microvilli as well as numerous dense bodies including premelanosomes and varied multilamellar structures. Ruthenium red labeling revealed the frequent basal localization of the tight junction. The cells were identified to be of rat RPE origin by their expression of the rat RPE marker RET-PE2 and their ability to phagocytose latex beads. While RPE-J cells are capable of sorting influenza and vesicular stomatitis virus to the apical and basal surfaces, respectively, the Na,K-ATPase is not polarized and the neural cell adhesion molecule, N-CAM, is localized exclusively to the lateral surface. In vivo the apical surface of RPE interacts with the adjacent neural retina and the Na,K-ATPase and N-CAM are both apical; the altered polarity of these two proteins in RPE-J cells may be a consequence of the absence of apical interaction with the neural retina in culture. Previous studies of RPE have been restricted to the use of primary cultures and the RPE-J cell line should prove an excellent model system for the study of the mechanisms determining the characteristic polarity and functions of the retinal pigment epithelium.

Replication-incompetent variants of the avian spleen necrosis virus (SNV) encoding cytoplasmic or nuclear-directed beta-galactosidase (beta-gal) have been used to trace the clonal growth of myocytes during left ventricular free-wall formation. Tubular-stage hearts were infected with a mixed suspension of both retroviruses and, after hatching, the progeny of marked cells in the ventricular wall were examined by X-gal histochemistry. When a small number of virions was introduced individual blue patches contained myocytes with only one label type (cytoplasmic or nuclear). These results confirmed our previous conclusion that each cluster or patch represents a single clone (Mikawa et al., 1992, Dev. Dynamics, 193:11-23). Each of these clones formed a clone-shaped patch which often extended through the entire thickness of the ventricular myocardium, but typically each patch was heterogeneous, containing a mixture of labeled and unlabeled cells. We then asked whether the two populations of myocytes in each patch were clonally related or generated from more than one progenitor. When hearts were infected with high titer viral suspensions many patches were observed in which cytoplasmic-tagged myocytes were intermingled with nuclear-tagged myocytes. Thus, the cone-shaped myocyte patches in the ventricular wall are polyclones derived from separate progenitors in the precardiac mesoderm. This finding led us to examine the separation of clonally related ventricular myocytes in the developing hearts. Embryos were infected with retroviral suspensions at varying stages of development and the resulting colonies examined after hatching.(ABSTRACT TRUNCATED AT 250 WORDS)

The distribution of sarcomeric myosin heavy chain (MyHC) has been examined immunocytochemically in the presumptive myocardial cells of chicken embryos (stages 6-10) prior to the onset of the heart beat. Embryos were stained with monoclonal antibody MF20, a reagent which recognizes all chicken sarcomeric MyHCs (Bader et al., 1982), and then examined both in whole mount by immunofluorescence and in semithin, plastic-embedded sections following immunoperoxidase labeling. We observed that myosin could be detected as early as stage 7 (0-2 pairs of somites) in 29% of the 31 embryos examined, and by stage 8 (4 pairs of somites) more than 80% of the embryos were MF20+. Every embryo with 5 pairs of somites (stage 8+) labeled strongly with MF20. Labeling was first detected at stage 7 to 7+ as a diffuse fluorescent signal within pleomorphic cells of the splanchnic mesoderm located in two crescent-shaped regions bordering each side of the anterior intestinal portal (AIP). With progressive development, the two crescent-shaped regions merged at the apex of the AIP, and as the two heart tubes began fusion at stage 9, the MyHC+ regions extended cranially and medially. By somite stages 9-10, the myosin-positive cells completely encircled the heart tube. From stages 7 to 9 the myosin signal had no sarcomeric distribution; i.e., there were no MyHC striations nor periodic repeats evident in the presumptive myocytes until late stage 9 and stage 10. Semithin sections revealed that myosin was first distributed in apical regions of the myocytes, adjacent to the pericardial coelom. The implications of these findings for myocyte determination, differentiation and morphogenesis are discussed.

Recruitment of various stem and progenitor cells is crucial for the regeneration of an injured organ. Levels of uric acid, one of the prototypical “alarm signals,” surge after ischemia-reperfusion injury. Exogenous uric acid rapidly mobilizes endothelial progenitor cells and hematopoietic stem cells and protects the kidney from ischemia. The relatively fast responses to uric acid suggest that preformed second messengers may be released from a storage pool. Here, it is reported that monosodium urate (MSU) results in exocytosis of Weibel-Palade bodies in vitro and in vivo , leading to the release of IL-8, von Willebrand factor, and angiopoietin 2 in the culture medium or circulation. Confocal and immunoelectron microscopy confirmed depletion of von Willebrand factor in MSU-treated aortic endothelial cells. Angiopoietin 2 alone induced exocytosis of Weibel-Palade bodies, mobilized hematopoietic stem cells and depleted splenic endothelial progenitor cells, partially reproducing the actions of MSU. In addition, pretreatment with angiopoietin 2 protected the kidneys from an ischemic insult, suggesting that the previously reported renoprotection conferred by MSU likely results from exocytosis of Weibel-Palade bodies. Furthermore, experiments with toll-like receptor 4 (TLR-4)–and TLR-2–deficient mice demonstrated that uric acid–induced exocytosis of Weibel-Palade bodies is mediated by TLR-4 and that uric acid–induced release of IL-8 requires both TLR-2 and TLR-4. In summary, these results suggest that exocytosis of Weibel-Palade bodies links postischemic repair with inflammation and mobilization of stem cells.

The mechanisms that mediate the establishment of totipotency during the egg-to-embryo transition in mammals remain poorly understood. However, it is clear that unique factors stored in the oocyte cytoplasm are crucial for orchestrating this complex cellular transition. The oocyte cytoplasmic lattices (CPLs) have long been predicted to function as a storage form for the maternal contribution of ribosomes to the early embryo. We recently demonstrated that the CPLs cannot be visualized in Padi6-/- oocytes and that Padi6-/-embryos arrest at the two-cell stage. Here, we present evidence further supporting the association of ribosomes with the CPLs by demonstrating that the sedimentation properties of the small ribosomal subunit protein, S6, are dramatically altered in Padi6-/- oocytes. We also show that the abundance and localization of ribosomal components is dramatically affected in Padi6-/- two-cell embryos and that de novo protein synthesis is also dysregulated in these embryos. Finally, we demonstrate that embryonic genome activation (EGA) is defective in Padi6-/- two-cell embryos. These results suggest that, in mammals, ribosomal components are stored in the oocyte CPLs and are required for protein translation during early development.

Chronic kidney diseases are accompanied by the accumulation of substances like asymmetric dimethylarginine, phenylacetic acid, homocysteine, and advanced glycation end products, known to either inhibit endothelial nitric oxide synthase (eNOS) or uncouple it, consequently limiting the amount of available nitric oxide (NO). Reduced bioavailability of NO induces endothelial dysfunction. An early loss of peritubular capillaries in tubulointerstitial fibrotic areas and injury to endothelial cells have been linked to progressive renal disease. Screening endothelial genes in cells treated with NOS inhibitors showed upregulation of collagen XVIII, a precursor of a potent antiangiogenic substance, endostatin. This finding was confirmed at the level of mRNA and protein expression. Tie-2 promoter-driven green fluorescent protein mice treated with nonhypertensinogenic doses of a NOS inhibitor exhibited upregulation of collagen XVIII/endostatin and rarefaction of capillary profiles. This was accompanied by the increased expression of transforming growth factor-β and connective tissue growth factor in the kidney. Occasional endothelial cells expressed both the marker of endothelial lineage (green fluorescent protein) and mesenchymal marker (α-smooth muscle actin or calponin). In vitro studies of endothelial cells treated with asymmetric dimethylarginine showed decreased expression of eNOS and Flk-1 and enhanced expression of calponin and fibronectin, additional markers of smooth muscle and mesenchymal cells. These cells overexpressed transforming growth factor-β and connective tissue growth factor, as well as endostatin. In conclusion, data presented here 1) ascribe to NO deficiency in endothelial cells the function of a profibrotic stimulus associated with the expression of an antiangiogenic fragment of collagen XVIII (endostatin) and 2) provide evidence of endothelial-mesenchymal transdifferentiation in the course of inhibition of NOS by a pathophysiologically important antagonist, asymmetric dimethylarginine. Both mechanisms may account for microvascular rarefaction.

Caveolae and their associated structural proteins, the caveolins, are specialized plasmalemmal microdomains involved in endocytosis and compartmentalization of cell signaling. We examined the expression and distribution of caveolae and caveolins in retinal pigment epithelium (RPE), which plays key roles in retinal support, visual cycle, and acts as the main barrier between blood and retina. Electron microscopic observation of rat RPE, in situ primary cultures of rat and human RPE and a rat RPE cell line (RPE-J) demonstrated in all cases the presence of caveolae in both apical and basolateral domains of the plasma membrane. Caveolae were rare in RPE in situ but were frequent in primary RPE cultures and in RPE-J cells, which correlated with increased levels in the expression of caveolin-1 and -2. The bipolar distribution of caveolae in RPE is striking, as all other epithelial cells examined to date (liver, kidney, thyroid, and intestinal) assemble caveolae only at the basolateral side. This might be related to the nonpolar distribution of both caveolin-1 and 2 in RPE because caveolin-2 is basolateral and caveolin-1 nonpolar in other epithelial cells. The bipolar localization of plasmalemmal caveolae in RPE cells may reflect specialized roles in signaling and trafficking important for visual function.

Despite growing evidence for a mitochondrial localization of nitric oxide (NO) synthase and a broadening spectrum of NO actions on mitochondrial respiration and apoptosis, the basis for interaction between the enzyme and the organelle remain obscure. Here we investigated mitochondrial localization of endothelial nitric oxide synthase (eNOS) in human umbilical vein endothelial cells and human embryonic kidney cells transfected or infected with eNOS expression vectors. Copurification of eNOS with mitochondria was observed in both human umbilical vein endothelial cells and eNOS-expressing human embryonic kidney cells. Immunodetectable eNOS was cleaved from mitochondria by proteinase K treatment, suggesting eNOS association with the outer mitochondrial membrane. Localization of eNOS to a proteinase K-cleavable site on the cytoplasmic face of the outer membrane was confirmed by immunogold labeling of non-permeabilized mitochondria. Markers for mitochondrial subfractions ruled out the possibility of eNOS association with an intramitochondrial site or inverted mitochondrial particles. Denaturation of eNOS did not attenuate association with mitochondria. Mutant eNOS lacking a pentabasic amino acid sequence within the autoinhibitory domain (residues 628-632 of the bovine eNOS) showed dramatically reduced binding to the mitochondrial but not to the plasma membrane, which was associated with increased oxygen consumption. Collectively, these findings argue in favor of eNOS localization to the outer mitochondrial membrane in endothelial cells and identify elements of a novel anchoring mechanism.

For people in a small EM lab, keeping stable stain solutions that don't accumulate precipitates over time can be a problem. Over the last 26 years, I have adapted the method originally described by Venable and Coggeshall (1) to address this need

A primary function of cadherins is to regulate cell adhesion. Here, we demonstrate a broader function of cadherins in the differentiation of specialized epithelial cell phenotypes. In situ, the rat retinal pigment epithelium (RPE) forms cell-cell contacts within its monolayer, and at the apical membrane with the neural retina; Na+, K(+)-ATPase and the membrane cytoskeleton are restricted to the apical membrane. In vitro, RPE cells (RPE-J cell line) express an endogenous cadherin, form adherens junctions and a tight monolayer, but Na+,K(+)-ATPase is localized to both apical and basal-lateral membranes. Expression of E-cadherin in RPE-J cells results in restriction and accumulation of both Na+,K(+)-ATPase and the membrane cytoskeleton at the lateral membrane; these changes correlate with the synthesis of a different ankyrin isoform. In contrast to both RPE in situ and RPE-J cells that do not form desmosomes, E-cadherin expression in RPE-J cells induces accumulation of desmoglein mRNA, and assembly of desmosome-keratin complexes at cell-cell contacts. These results demonstrate that cadherins directly affect epithelial cell phenotype by remodeling the distributions of constitutively expressed proteins and by induced accumulation of specific proteins, which together lead to the generation of structurally and functionally distinct epithelial cell types.

We have used light and electron microscopy to analyze endocytic trafficking pathways. In one set of studies, we have used fluorescently labeled antibodies to trace an endocytic pathway from the cell surface to the trans- Golgi network (TGN). Cells were transfected with a construct consisting of the transmembrane and cytoplasmic domains of TGN38 and the extracellular domain of Tac. TGN38 is predominantly in the TGN, but a small fraction is found on the cell surface. We used FITC-labeled anti-Tac monoclonal IgG to analyze the pathway from the surface to the TGN. We compared the distribution of internalized Tac-TGN38 to internalized transferrin. We found that most Tac-TGN38 enters the same early endosomes as transferrin. Furthermore, most Tac-TGN38 returns to the cell surface from the endocytic recycling compartment (ERC) at the same rate as transferrin. However, on each pass through the cell approximately 18% of Tac-TGN is retained, and this Tac-TGN38 is delivered to the TGN.

In situ, ultrastructural measurements of diameters of contractile filaments in skeletal and heart muscle differ considerably from those previously reported. Past measurements have been made in thin, transverse epoxy sections that were non-specifically stained with heavy metal salts to overcome background scattering of epoxy polymer. In our images from transverse de-embedded sections, the hexagonal lattice has some considerable differences from that seen in epoxy sections. Muscle samples from rat atrium and frog sartorius were fixed, dehydrated, embedded in polyethylene glycol, and sectioned. Sections were de-embedded in graded polyethylene glycol/ethanol, mounted on coated grids, critical point dried, and viewed in the electron microscope without staining. The backbone diameters of thick filaments were measured in the M band region and have an average value, after correction for shrinkage, of 25 nm. Thin filament diameters range from 6.5–9.5 nm. In regions of overlap of thin and thick filaments, the thick filament profiles varied from circular to asymmetric; diameters range up to 36 nm and yield eccentricity ratios varying from 1.5 to 1.0 (circular profiles). Portions of thick filaments touch or partially envelope neighboring thin filaments. The relative contributions of cytoskeletal components to these images of overlap regions remains to be determined, but the backbone diameters in glycerinated frog sartorius are not significantly different from control samples. The present results are consistent with those reported for rotary shadowed thick filaments; from recent experiments in muscles whose myofilament lattice is osmotically compressed; and with estimates of A band mass. This lattice geometry yields relatively low surface-to-surface distances between filaments. Steric considerations and their implications for cross bridge theory are discussed.

The extracellular matrix of heart muscle contains a considerable variety of structures. We have systematically studied the morphology of these structures using several methods of fixation and microscopy. Endomysial connections between cells are comprised of struts of collagen [1] as well as combinations of elastin fibers, collagen fibers, and microfibrils. The rest of the extracellular matrix is filled with a polyanionic lattice of unit collagen fibrils, microthreads, and granules. In the course of these investigations, we have observed regions of structural continuity across the sarcolemma, from endomysial collagen struts to Z-bands. We have also correlated the mechanical resistance to stretch with orientation of epimysial collagen fibers and sarcomere lengths in living as well as fixed rat papillary muscles. Our observations suggest that the extracellular skeletal framework plays an important role in normal cardiac function.

Advances in Immunochemistry allow for labelling of various tissue components with highly specific antibodies, that in turn may be labelled with marker substances such as flourescein for light microscopy, or any of a number of electron-dense substances, such as ferritin and colloldal gold, for localization with scanning and transmission electron miscroscopy. The methods used for transmission electron microscopy are designed to minimize damage to antigenic sites while preserving ultrastructure. The methods include embedding of the tissue in resins with low-temperature curing characteristics such as Polyethylene glycol (PEG) or Lowicryl K4M (Chemische Werke Lowl, F.R.G.). The tissue is embedded, sectioned, and mounted on grids. The Immuno-reactions are then carried out on these mounted sections.