Northwestern University Feinberg School of Medicine

Kathleen Green Lab

The Importance of Cell Biology: Cells and Molecules are at the Root of Tissue Functions

Figure 1

To understand tissue morphogenesis and disease pathogenesis, ultimately we must understand what happens at the cellular and molecular levels.   To do this, we use a number of techniques.  To identify new protein-protein interactions important for establishing the machinery through which junctions carry out structural and signaling functions, we use in vitro biochemistry and a variety of protein interaction screens, including recently emerging Bio-ID proteomic approaches that allow for mapping nearest neighbors in cells and tissues in situ. How these protein interactions are regulated by post-translational modifications, including phosphorylation and protein methylation is also being uncovered through the use of mass spectrometry approaches.

To look at the importance of these protein interactions in cells, state-of-the-art optical imaging techniques are being employed. Fluorescently-tagged wild type and mutant desmosome and adherens junction molecules are tracked during intercellular junction assembly, to determine how they segregate themselves into distinct plasma membrane domains.   Live cell imaging and biochemical approaches are also being used to elucidate mechanisms by which the transmembrane and plaque compartments are trafficked through distinct MT- and actin-dependent paths to the site of junction assembly at the membrane.  Taking junction-cytoskeletal dynamics into 3D, we use state-of-the-art imaging capabilities to track protein and cell dynamics during epidermal morphogenesis in human organotypic epidermal cultures.

Our recent work has revealed the importance of specific kinesin motors in delivering desmosomal cadherins to the plasma membrane, and show that kinesin deficiency disrupts intercellular adhesion by interfering with desmosome function, included here is a model of desmosomal cadherin intracellular trafficking to the plasma membrane taken from our recent publication (Figure 1. O. Nekrasova et al. 2012. J Cell Biol. 195:1185).  Desmoglein 2 (Dsg2) and Desmocollin 2 (Dsc2) traffic in separate vesicular compartments that are transported using distinct motors, kinesin-1 and kinesin-2, respectively (IF = intermediate filament).

Figure 1

Determining the Importance of Desmosomes in Inherited Skin and Heart Diseases and Cancer

Figure 2. Figure 7. Immunofluorescence analysis of skin sections from control and SAM syndrome affected individuals. Cytoplasmic accumulation of DSG1 or loss of DSG1 staining in patients carrying mutations c.49-1G>A (Family A) and c.1861delG (Family B), respectively, compared with the plasma membrane localization in control samples. DSP staining is retained in both SAM syndrome individuals. Samuelov, L., et al. Nature Genetics. 2006; 45:1244-8.

Desmosome molecules are targets in inherited, autoimmune and bacterial toxin-mediated disease that can lead to a variety of skin disorders, allergies and human cardiomyopathies ranging in severity from mild to lethal.  For example, in collaboration with Eli Sprecher (Tel Aviv) we reported a new syndrome in which patients have severe dermatitis, multiple allergies, and metabolic wasting (SAM) due to complete loss of Dsg1 or a mutation that results in defective trafficking (Figure 2).  Interestingly, cells isolated from the epidermis of these patients exhibit keratinocyte-autonomous production of cytokines, raising the possibility that reduced Dsg1 function in keratinocytes can affect paracrine signaling that could contribute to systemic influences on the immune system.  Efforts are underway to elucidate the signaling pathways involved, and to determine the extent to which previously reported regulation of MAPK signaling may contribute to the observed effects.

Figure 2. Figure 7. Immunofluorescence analysis of skin sections from control and SAM syndrome affected individuals. Cytoplasmic accumulation of DSG1 or loss of DSG1 staining in patients carrying mutations c.49-1G>A (Family A) and c.1861delG (Family B), respectively, compared with the plasma membrane localization in control samples. DSP staining is retained in both SAM syndrome individuals. Samuelov, L., et al. Nature Genetics. 2006; 45:1244-8.

Figure 3. 3D Keratinocyte-Melanocyte co-culture. A) Gross morphology of control and melanocyte-containing (note dark pigment) cultures on screens used for lifting to air-medium interface. B) Sections from paraffin-embedded control and Dsg1 knock down cultures stained for Dsg1 (green), S100B to mark melanocytes (red; arrows), and DAPI (blue).

Desmoglein-dependent regulation of paracrine signaling may also be important in cancer.  We have observed elevated expression of pro-tumorigenic interleukins, including those known to promote melanoma cell invasion. Further, co-culture of melanocytes with Dsg1-silenced keratinocytes induced production of pro-tumorigenic melanocyte-derived cytokines. We hypothesize that loss of Dsg1 function in melanoma-associated keratinocytes promotes melanoma progression through pro-tumorigenic paracrine signaling. Interestingly, normal human melanocytes co-cultured in 3D with Dsg1-deficient keratinocytes are not as effectively retained in the basal layers and also exhibit signs of increased proliferation, indicating a possible role for Dsg1 in controlling normal melanocyte behavior (Figure 3).  We will incorporate melanoma cells from different staged tumors in similar co-cultures to address whether Dsg1 is important for suppressing their invasion.

Figure 3. 3D Keratinocyte-Melanocyte co-culture. A) Gross morphology of control and melanocyte-containing (note dark pigment) cultures on screens used for lifting to air-medium interface. B) Sections from paraffin-embedded control and Dsg1 knock down cultures stained for Dsg1 (green), S100B to mark melanocytes (red; arrows), and DAPI (blue).

Figure 4

Desmosome mutations have also been linked with a high prevalence to the cardiac disease, Arrhythmogenic Right Ventricular Cardiomyopathy (ARVC), so much so that this disease is often referred to as the disease of the desmosome. Our lab is using in vitro biochemical and cell biological analysis to understand how specific human gene mutations affect protein function, as described below and shown in the associated figure (Figure 4). This figure illustrates that cardiac expression of the cardiac disease-related mutation in desmoplakin (R2834H) can result in cardiac hypertrophy as demonstrated by a change in the gross morphology of mouse hearts and a significant increase in the heart weight:body weight ratio of the animals compared to the over-expression of wild-type desmoplakin. Our recent work suggests that this mutation interferes with arginine methylation of the desmoplakin C-terminus, a post-translational modification required for optimal GSK3beta-dependent processive phosphorylation necessary for normal desmoplakin dynamics (L. Albrecht et al. 2015. J Cell Biol. 208:597-612).

Figure 4