Browsing by Subject "Epidermis"
- Results Per Page
- Sort Options
Item Open Access Elaborating the Desmosome Proteome: Insights into Novel Mechanisms Essential for Regulating Epidermal Integrity and Homeostasis(2020) Badu-Nkansah, Kwabena AgyemanDesmosomes are a class of cell-cell adhesions whose primary physiological function is to maintain the mechanical integrity of tissues. Morphologically, desmosomes are very distinct protein complexes. They are dense pairs of plaques that straddle the interface between two plasma membranes of adjacent cells. In the extracellular space between each plaque are series of desmosome receptors that mediate the cell-cell attachment events and drive cellular cohesion throughout tissues. Emanating from the intracellular face of desmosomes are networks of intermediate filaments to which desmosome plaques are anchored. It is through this molecular chain that desmosomes traditionally bear mechanical load and promote tissue integrity. Molecular roles of the core components sufficient for completing this chain have been well described. However, insights from desmosome pathologies, genetic interactions, and biochemical fractionation have introduced perspectives that strongly suggest that desmosome composition and function extends beyond what is currently known. It is within this general context that I began my exploration into desmosome biology and employed global approaches towards uncovering a novel proteome that revealed unexpected interactions important for tissue function.
First, we used targeted proximity labeling approaches to elaborate the desmosome proteome in epidermal keratinocytes. Quantitative mass spectrometry analysis uncovered a diverse array of new constituents with broad molecular functions. We validated a number of novel desmosome-associated proteins and found that many are membrane proximal proteins that show a dependence on functional desmosomes for their cortical localization. We further explored the mechanism of localization and function of two adaptor proteins enriched in the desmosome proteome, Crk and CrkL. Epidermal deletion of both Crk and CrkL resulted in perinatal lethality with defects in desmosome morphology and keratin organization, thus demonstrating the utility of this dataset in identifying novel proteins required for desmosome-dependent epidermal integrity.
Desmosomes role in mechanical integrity is paramount in the epidermis as skin-blistering pathologies represent one of the primary hallmarks of diseases caused by desmosome disruption. Pemphigus Vulgaris (PV) is a series of autoimmune syndromes where patients produce autoantibodies that target desmosome receptors, desmoglein 3 and desmoglein 1. PV is characterized by weakening cell adhesions that result in widespread epidermal cell separations. While pathologies that characterize PV have been long documented, molecular mechanisms that drive initial desmosome disruption are still not fully understood. We sought to address initial molecular signaling events induced by PV antibodies using targeted proteomic analysis to identify early phosphorylation events. We performed time course phosphoproteomic analysis on keratinocytes early after exposure to PV antibody, AK23. With this approach we observed large coverage of the keratinocyte phosphoproteome that included significant shifts of abundant phosphorylation events after AK23 treatment. To identify the functional relevance of novel phosphorylation events, we followed the localization of constructs containing tagged protein candidates harboring phosphomimetic or non-phosphorylatable mutations. Altogether, these results provide an initial analysis of global phosphorylation events that occur in keratinocytes during the early stages of PV.
Finally, we sought to understand what was an unusual enrichment of RNA-binding proteins in our desmosome proteome. Amongst enriched protein classes were regulators of protein translation. Using immunofluorescent approaches, we were able to visualize distinct desmosome localization of representative members of protein translation machinery as well as assembled ribosomes. Considering this unusual abundance, I was interested in finding out the types of RNAs associated with these desmosome-associated RBPs. To do this we combined our proteomic approach with RNA sequencing and developed new techniques to enrich for specific RNA subpopulations localized at desmosomes. By probing for candidates of interest from this list I was able to observe enrichment of specific transcripts at desmosomes, suggesting an intimate link between RNA regulation and cell-cell adhesion. Taken together, these studies represent the first comprehensive proteomic analyses of desmosome adhesions and highlight novel desmosome interactions that may broaden mechanistic roles for desmosomes in epithelial biology.
Item Open Access Investigating Dynamics of Tissue Regeneration via Live Imaging of Zebrafish Scales(2019) Cox, BenRegeneration occurs throughout the animal kingdom and is a well-studied
feature of many model organisms, yet the field lacks a fundamental understanding of
the real-time dynamics of cell behavior during regeneration. I discuss how existing
knowledge of regeneration may be used to inform efforts to translate these remarkable
feats of animals to human regeneration and present research that uses live imaging to
improve understanding of cell origins and diversification during regeneration in the
scale, focusing specifically on osteoblasts the matrix-depositing cells that divide and heal
bone injuries. I developed an imaging platform to monitor and quantify individual and
collective behaviors of osteoblasts in adult zebrafish scales. I show that a founder pool
of osteoblasts emerges through de novo differentiation within one day of scale plucking,
then diversifies across the primordium by two days after injury, with region-specific
changes in proliferation, cell shape, and cell death rates coincident with acquisition of
mature scale morphology. I also demonstrate a role for Fgf signaling in scale
regeneration and present tools for high resolution imaging studies of basal epidermal
cells during skin and scale injury. These findings demonstrate the value of live imaging
in revealing novel biology and gaining a more complete picture of the many complex
processes that must be elegantly choreographed to achieve tissue regeneration.
Item Open Access Pairing of competitive and topologically distinct regulatory modules enhances patterned gene expression.(Mol Syst Biol, 2008) Yanai, Itai; Baugh, L Ryan; Smith, Jessica J; Roehrig, Casey; Shen-Orr, Shai S; Claggett, Julia M; Hill, Andrew A; Slonim, Donna K; Hunter, Craig PBiological networks are inherently modular, yet little is known about how modules are assembled to enable coordinated and complex functions. We used RNAi and time series, whole-genome microarray analyses to systematically perturb and characterize components of a Caenorhabditis elegans lineage-specific transcriptional regulatory network. These data are supported by selected reporter gene analyses and comprehensive yeast one-hybrid and promoter sequence analyses. Based on these results, we define and characterize two modules composed of muscle- and epidermal-specifying transcription factors that function together within a single cell lineage to robustly specify multiple cell types. The expression of these two modules, although positively regulated by a common factor, is reliably segregated among daughter cells. Our analyses indicate that these modules repress each other, and we propose that this cross-inhibition coupled with their relative time of induction function to enhance the initial asymmetry in their expression patterns, thus leading to the observed invariant gene expression patterns and cell lineage. The coupling of asynchronous and topologically distinct modules may be a general principle of module assembly that functions to potentiate genetic switches.Item Open Access Regulated spindle orientation buffers tissue growth in the epidermis.(eLife, 2019-10) Morrow, Angel; Underwood, Julie; Seldin, Lindsey; Hinnant, Taylor; Lechler, TerryTissue homeostasis requires a balance between progenitor cell proliferation and loss. Mechanisms that maintain this robust balance are needed to avoid tissue loss or overgrowth. Here we demonstrate that regulation of spindle orientation/asymmetric cell divisions is one mechanism that is used to buffer changes in proliferation and tissue turnover in mammalian skin. Genetic and pharmacologic experiments demonstrate that asymmetric cell divisions were increased in hyperproliferative conditions and decreased under hypoproliferative conditions. Further, active K-Ras also increased the frequency of asymmetric cell divisions. Disruption of spindle orientation in combination with constitutively active K-Ras resulted in massive tissue overgrowth. Together, these data highlight the essential roles of spindle orientation in buffering tissue homeostasis in response to perturbations.Item Open Access Regulation of Asymmetric Cell Divisions in the Developing Epidermis(2012) Poulson, NicholasDuring development, oriented cell divisions are crucial for correctly organizing and shaping a tissue. Mitotic spindle orientation can be coupled with cell fate decisions to provide cellular diversity through asymmetric cell divisions (ACDs), in which the division of a progenitor cell results in two daughters with different cell fates. Proper tissue morphogenesis relies on the coupling of these two phenomena being highly regulated. The development of the mouse epidermis provides a powerful system in which to study the many levels that regulate ACDs. Within the basal layer of the epidermis, both symmetric and asymmetric cell divisions occur. While symmetric divisions allow for an increase in surface area and progenitor cell number, asymmetric divisions drive the stratification of the epidermis, directly contributing additional cell layers (Lechler and Fuchs 2005; Poulson and Lechler 2010; Williams, Beronja et al. 2011).
Utilizing genetic lineage tracing to label individual basal cells I show that individual basal cells can undergo both symmetric and asymmetric divisions. Therefore, the balance of symmetric:asymmetric divisions is provided by the sum of individual cells' choices. In addition, I define two control points for determining a cell's mode of division. First is the expression of the mInscuteable gene, which is sufficient to drive ACDs. However, there is robust control of division orientation as excessive ACDs are prevented by a change in the localization of NuMA, an effector of spindle orientation. Finally, I show that p63, a transcriptional regulator of stratification, does not control either of these processes, rather it controls ACD indirectly by promoting cell polarity.
Given the robust control on NuMA localization to prevent excess ACDs, I sought to determine how targeting of NuMA to the cortex is regulated. First, I determined which regions within the protein were necessary and sufficient for cortical localization. NuMA is a large coiled- coil protein that binds many factors important for ACDs, which include but are not limited to: microtubules, 4.1, and LGN. Interestingly, while the LGN binding domain was necessary, it was not sufficient for proper NuMA localization at the cortex. However, a fragment of NuMA containing both the 4.1 and LGN binding domains was able to localize to the cortex. Additionally, the NuMA-binding domain of 4.1 was able to specifically disrupt NuMA localization at the cortex. These data suggested an important role for a NuMA-4.1 interaction at the cortex. While the 4.1 binding domain was not necessary for the cortical localization of NuMA, it was important for the overall stability of NuMA at the cortex. I hypothesize that 4.1 acts to anchor/stabilize NuMA at the cortex to provide resistance against pulling forces on the mitotic spindle to ensure proper spindle orientation.
Finally, to determine if post-translational modifications of NuMA could regulate its localization I tested the importance of a conserved Cdk-1 phosphorylation site. Interestingly, a non-phosphorylatable form of NuMA localized predominately to the cortex while the phosphomimetic protein localized strongly to spindle poles. In agreement with these data, use of a CDK-1 inhibitor was able to enhance the cortical localization of NuMA. Unexpectedly, the non-phosphorylatable form of NuMA did not require LGN to localize to the cortex. Additionally, restoration of cortical localization of the phosphomimetic form of NuMA was accomplished by the overexpression of either LGN or 4.1. Thus, phosphorylation of NuMA may alter its overall affinity for the cortex.
Overall, my studies highlight two important regulatory mechanisms controlling asymmetric cell division in the epidermis. Additionally, I show a novel role for the interaction between NuMA and 4.1 in providing stability at the cortex. This will ultimately provide a framework for analysis of how external cues control the important choice between asymmetric and symmetric cell divisions.
Item Open Access RNA-Seq and ChIP-Seq reveal SQSTM1/p62 as a key mediator of JunB suppression of NF-κB-dependent inflammation.(J Invest Dermatol, 2015-04) Zhang, Xiaoling; Jin, Jane Y; Wu, Joseph; Qin, Xiaoxia; Streilein, Robert; Hall, Russell P; Zhang, Jennifer YMice with epidermal deletion of JunB transcription factor displayed a psoriasis-like inflammation. The relevance of these findings to humans and the mechanisms mediating JunB function are not fully understood. Here we demonstrate that impaired JunB function via gene silencing or overexpression of a dominant negative mutant increased human keratinocyte cell proliferation but decreased cell barrier function. RNA-seq revealed over 500 genes affected by JunB loss of function, which included the upregulation of an array of proinflammatory molecules relevant to psoriasis. Among these were tumor necrosis factor α (TNFα), CCL2, CXCL10, IL6R, and SQSTM1, an adaptor protein involved in nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) activation. Chromatin immunoprecipitation (ChIP)-Seq and gene reporter analyses showed that JunB directly suppressed SQSTM1 by binding to a consensus AP-1 cis element located around 2 kb upstream of SQSTM1-transcription start site. Similar to JunB loss of function, SQSTM1-overexpression induced TNFα, CCL2, and CXCL10. Conversely, NF-κB inhibition genetically with a mutant IκBα or pharmacologically with pyrrolidine dithiocarbamate (PDTC) prevented cytokine, but not IL6R, induction by JunB deficiency. Taken together, our findings indicate that JunB controls epidermal growth, barrier formation, and proinflammatory responses through direct and indirect mechanisms, pinpointing SQSTM1 as a key mediator of JunB suppression of NF-κB-dependent inflammation.Item Open Access Spindle Orientation Coordinates Cell Fate Decisions During Epidermal Morphogenesis(2021) Moreci, Mary Rebecca StockdaleAsymmetric cell divisions (ACDs) drive cell fate specification and the formation of complex tissue architecture. This often requires orienting the mitotic spindle to position daughter cells and/or segregate cell fate determinants. However, the mechanisms that properly position the mitotic spindle and link spindle orientation to cell fate specification are not fully understood. During mouse embryogenesis, oriented divisions drive epidermal morphogenesis. Disrupting oriented divisions results in differentiation defects, loss of barrier function, and lethality. ACDs have also been observed during hair follicle morphogenesis and have been suggested to produce specific populations of hair follicle cells, though this has not been directly tested. In the developing epidermis, spindle orientation requires a conserved cortical protein complex of LGN/NuMA/dynein-dynactin. This complex is thought to function by generating pulling forces on astral microtubules. However, the factors that regulate astral microtubule dynamics and their association with this cortical complex have not been well studied. In this work, I explored the role of the microtubule catastrophe factor KIF18B in regulating microtubule dynamics to promote spindle orientation in keratinocytes. Utilizing two individually isolated control and KIF18B knockout (KIF18BKO) cell lines, I demonstrated that KIF18B is required for spindle alignment in cultured keratinocytes. Mitotic spindles in KIF18BKO cells exhibited an increase in both the length and number of astral microtubules. Microtubules more frequently touched the cortex in KO cells than in control cells. Furthermore, live-imaging revealed that loss of KIF18B dramatically altered microtubule dynamics; astral microtubules of KIF18BKO cells showed an increase in growth lifetime and growth displacement compared to controls. KIF18B’s regulation of microtubules was restricted to the astral microtubules, which is of note, as these are the microtubules that interact with the cell cortex as well as the spindle orientation machinery. I propose that KIF18B promotes astral microtubule catastrophe, which is essential to maintain proper microtubule dynamics and orient the mitotic spindle. Unexpectedly, I discovered that KIF18B accumulates at the cell cortex during mitosis, colocalizing with the conserved spindle orientation machinery. This cortical localization has not been previously reported. KIF18B cortical localization mimicked that of NuMA. During metaphase, KIF18B polarized to one side of the cell cortex. However, during anaphase, KIF18B became bipolar and expanded along the cell cortex. This localization pattern was NuMA-dependent in both metaphase and anaphase and did not require microtubules. In vivo I found that KIF18B was required for oriented cell divisions within the hair placode, the first stage of hair follicle morphogenesis, but was not essential in the interfollicular epidermis. Disrupting spindle orientation in the placode, using mutations in either KIF18B or NuMA, resulted in aberrant expression of Sox9, which serves as a cell fate marker of the inner region of adult hair follicle cells. Additionally, I showed that treatment of mice with Wnt inhibitors phenocopied the abnormal Sox9 expression seen in ACD mutants. These data lead me to hypothesize that asymmetric segregation of Wnt regulators plays a role in asymmetric cell division of basal placode cells. My data functionally link spindle orientation to cell fate decisions during hair follicle morphogenesis for the first time. Taken together, my data demonstrate a role for regulated microtubule dynamics in spindle orientation in epidermal cells. My work also highlights the importance of spindle orientation during asymmetric cell division to dictate cell fate specification.
Item Open Access The Role of Spindle Orientation in Epidermal Development and Homeostasis(2015) Seldin, LindseyRobust regulation of spindle orientation is essential for driving asymmetric cell divisions (ACDs), which generate cellular diversity within a tissue. During the development of the multilayered mammalian epidermis, mitotic spindle orientation in the proliferative basal cells is crucial not only for dictating daughter cell fate but also for initiating stratification of the entire tissue. A conserved protein complex, including LGN, Nuclear mitotic apparatus (NuMA) and dynein/dynactin, plays a key role in establishing proper spindle orientation during ACDs. Two of these proteins, NuMA and dynein, interact directly with astral microtubules (MTs) that emanate from the mitotic spindle. While the contribution of these MT-binding interactions to spindle orientation remains unclear, these implicate apical NuMA and dynein as strong candidates for the machinery required to transduce pulling forces onto the spindle to drive perpendicular spindle orientation.
In my work, I first investigated the requirements for the cortical recruitment of NuMA and dynein, which had never been thoroughly addressed. I revealed that NuMA is required to recruit the dynein/dynactin complex to the cell cortex of cultured epidermal cells. In addition, I found that interaction with LGN is necessary but not sufficient for cortical NuMA recruitment. This led me to examine the role of additional NuMA-interacting proteins in spindle orientation. Notably, I identified a role for the 4.1 protein family in stabilizing NuMA's association with the cell cortex using a FRAP (fluorescence recovery after photobleaching)-based approach. I also showed that NuMA's spindle orientation activity is perturbed in the absence of 4.1 interactions. This effect was demonstrated in culture using both a cortical NuMA/spindle alignment assay as well as a cell stretch assay. Interestingly, I also noted a significant increase in cortical NuMA localization as cells enter anaphase. I found that inhibition of Cdk1 or mutation of a single residue on NuMA mimics this effect. I also revealed that this anaphase localization is independent of LGN and 4.1 interactions, thus revealing two independent mechanisms responsible for NuMA cortical recruitment at different stages of mitosis.
After gaining a deeper understanding of how NuMA is recruited and stabilized at the cell cortex, I then sought to investigate how cortical NuMA functions during spindle orientation. NuMA contains binding domains in its N- and C-termini that facilitate its interactions with the molecular motor dynein and MTs, respectively. In addition to its known role in recruiting dynein, I was interested in determining whether NuMA's ability to interact directly with MTs was critical for its function in spindle orientation. Surprisingly, I revealed that direct interactions between NuMA and MTs are required for spindle orientation in cultured keratinocytes. I also discovered that NuMA can specifically interact with MT ends and remain attached to depolymerizing MTs. To test the role of NuMA/MT interactions in vivo, I generated mice with an epidermal-specific in-frame deletion of the NuMA MT-binding domain. I determined that this deletion causes randomization of spindle orientation in vivo, resulting in defective epidermal differentiation and barrier formation, as well as neonatal lethality. In addition, conditional deletion of the NuMA MT-binding domain in adult mice results in severe hair growth defects. I found that NuMA is required for proper spindle positioning in hair follicle matrix cells and that differentiation of matrix-derived progeny is disrupted when NuMA is mutated, thus revealing an essential role for spindle orientation in hair morphogenesis. Finally, I discovered hyperproliferative regions in the interfollicular epidermis of these adult mutant mice, which is consistent with a loss of ACDs and perturbed differentiation. Based on these data, I propose a novel mechanism for force generation during spindle positioning whereby cortically-tethered NuMA plays a critical dynein-independent role in coupling MT depolymerization energy with cortical tethering to promote robust spindle orientation accuracy.
Taken together, my work highlights the complexity of NuMA localization and demonstrates the importance of NuMA cortical stability for productive force generation during spindle orientation. In addition, my findings validate the direct role of NuMA in spindle positioning and reveal that spindle orientation is used reiteratively in multiple distinct cell populations during epidermal morphogenesis and homeostasis.
Item Open Access Transient Receptor Potential Vanilloid 4 Ion Channel Functions as a Pruriceptor in Epidermal Keratinocytes to Evoke Histaminergic Itch.(J Biol Chem, 2016-05-06) Chen, Yong; Fang, Quan; Wang, Zilong; Zhang, Jennifer Y; MacLeod, Amanda S; Hall, Russell P; Liedtke, Wolfgang BTRPV4 ion channels function in epidermal keratinocytes and in innervating sensory neurons; however, the contribution of the channel in either cell to neurosensory function remains to be elucidated. We recently reported TRPV4 as a critical component of the keratinocyte machinery that responds to ultraviolet B (UVB) and functions critically to convert the keratinocyte into a pain-generator cell after excess UVB exposure. One key mechanism in keratinocytes was increased expression and secretion of endothelin-1, which is also a known pruritogen. Here we address the question of whether TRPV4 in skin keratinocytes functions in itch, as a particular form of "forefront" signaling in non-neural cells. Our results support this novel concept based on attenuated scratching behavior in response to histaminergic (histamine, compound 48/80, endothelin-1), not non-histaminergic (chloroquine) pruritogens in Trpv4 keratinocyte-specific and inducible knock-out mice. We demonstrate that keratinocytes rely on TRPV4 for calcium influx in response to histaminergic pruritogens. TRPV4 activation in keratinocytes evokes phosphorylation of mitogen-activated protein kinase, ERK, for histaminergic pruritogens. This finding is relevant because we observed robust anti-pruritic effects with topical applications of selective inhibitors for TRPV4 and also for MEK, the kinase upstream of ERK, suggesting that calcium influx via TRPV4 in keratinocytes leads to ERK-phosphorylation, which in turn rapidly converts the keratinocyte into an organismal itch-generator cell. In support of this concept we found that scratching behavior, evoked by direct intradermal activation of TRPV4, was critically dependent on TRPV4 expression in keratinocytes. Thus, TRPV4 functions as a pruriceptor-TRP in skin keratinocytes in histaminergic itch, a novel basic concept with translational-medical relevance.