Browsing by Author "Lechler, Terry"

Desmosomes 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.

Desmosomes are cell-cell adhesion complexes that provide tissues with mechanical integrity and their disruption results in severe blistering in the epidermis as well as cardiomyopathies. In this work, we sought to explore the various ways through which desmosomes function in keratinocytes to maintain connections between cells and promote cell sheet cohesion. Our approach examined both their canonical interactions with the intermediate filament cytoskeleton and a novel noncanonical association with RNA-induced silencing complexes.Keratin intermediate filaments form dynamic polymer networks that organize in specific ways dependent on the cell type, the stage of the cell cycle, and the state of the cell. In differentiated cells of the epidermis, they are organized by desmosomes, which provide essential mechanical integrity to this tissue. Despite this, we know little about how keratin organization is controlled and whether desmosomes locally regulate keratin dynamics or merely bind preassembled filaments. Ndel1 is a desmosome-associated protein in the differentiated epidermis that was previously described as being able to promote the assemble of neurofilaments, another class of intermediate filaments, although its function at desmosomes has not been examined. Here, we show that Ndel1 binds directly to keratin subunits through a motif conserved in all intermediate filament proteins. Further, Ndel1 was necessary for robust desmosome-keratin association and was sufficient to reorganize keratins at distinct cellular sites. Lis1, a Ndel1 binding protein, was required for desmosomal localization of Ndel1, but not for its effects on keratin filaments. Finally, we use mouse genetics to demonstrate that loss of Ndel1 results in desmosome defects in the epidermis. Our data thus identify Ndel1 as a desmosome-associated protein that promotes local assembly/reorganization of keratin filaments and is essential for robust desmosome formation. In addition to their canonical adhesive function, desmosomes have many emerging non-canonical functions such as regulating diverse signaling pathways and integrating diverse cytoskeletal networks in keratinocytes. Here we reveal a surprising recruitment of the RNA-induced silencing complex (RISC) to the cell cortex of keratinocytes. Using mutant cell lines, we find that desmosomes, but not adherens junctions, are required for the cortical localization of RISCs. We then identified the RISC-associated mRNAs specifically localized to the cortex using CLEAR-CLIP analysis which showed an enrichment for genes involved in wound healing and cell adhesion. Wound healing assays conducted both in vitro and in vivo revealed that RISCs become depleted from the cell cortex of keratinocytes near the site of injury. Further, perturbation of desmosomal adhesion using antibodies that target the desmosomes, which recapitulates the autoimmune disease pemphigus vulgaris, results in a similar loss of cortical Ago2. Our results define a new function for desmosomes in translational regulation and suggest that they may act as sensors of adhesion integrity which can directly influence the cell’s translatome to quickly restore homeostasis when it is disrupted. Although it is well established that desmosomes are important for the mechanical integrity of some tissues, it is only recently that we have come to understand the diversity of cellular processes they coordinate. Here we propose a mechanism through which desmosomes play a more active role in the organization of intermediate filaments than previously thought. Additionally, we demonstrate that they are involved in translational regulation via recruitment of RISCs and show that this population dynamically responds upon injury. Future investigations can further elucidate the role of the RISCs during wounding by probing the changes to the proteome shortly after wounding to determine the contributions of RISC-bound mRNA to recovery.

Microtubules play numerous roles in diverse cellular processes, including cell migration, intracellular trafficking, and chromosome segregation during cell division. Decades of research have defined many of the biochemical and biophysical properties of microtubules and how microtubule organization is controlled in proliferative cells. Diverse cell types rearrange their microtubules to form noncentrosomal microtubule arrays as they differentiate. However, little is known about the mechanisms regulating noncentrosomal microtubule array formation in many tissues, particularly in mammals. Because the mechanisms regulating formation of these arrays is largely mysterious, one of the key challenges in the field has been a lack of viable systems to specifically perturb microtubule organization in differentiated cells in vivo. I have used the mammalian epidermis and small intestine, two distinct types of epithelia, to investigate the formation and function of noncentrosomal microtubules.

In the mammalian epidermis, where differentiation is accompanied by a reorganization of microtubules from radial arrays, organized by the centrosome, to cortical arrays, I have uncovered a mechanism that links centrosome inactivation to cell-cycle exit. By purifying centrosomes from proliferative and differentiated keratinocytes, I demonstrate for the first time that keratinocyte differentiation induces an inherent inactivation of the centrosome. This is driven, in part, by the specific delocalization of a pool of the gamma-tubulin ring complex (gamma-TuRC) that is bound by the accessory factor Nedd1. I show that there are two distinct gamma-tubulin ring complexes in keratinocytes: one bound by Nedd1 and one bound by CDK5RAP2. Through both in vitro microtubule nucleation assays and artificial targeting of gamma-TuRCs in cells, I show that CDK5RAP2 can stimulate gamma-TuRC-mediated microtubule nucleation but Nedd1 cannot. I propose that Nedd1/gamma-TuRCs are, instead, critical for microtubule anchoring at the centrosome. To link differentiation status to centrosome inactivation, I show that, unexpectedly, differentiation per se does not induce Nedd1gamma-TuRC delocalization. Instead, cell cycle exit, which normally accompanies differentiation, triggers delocalization of gamma-TuRCs to shift microtubule organizing activity away from the centrosome. This work has identified for the first time the existence of distinct gamma-TuRCs within the cells and demonstrates that gamma-TuRC activity can be modified through binding of distinct accessory factors. Extending these findings to differentiation-induced microtubule reorganization, this work identifies a mechanism that couples entry into a quiescent state in post-mitotic cells to centrosome activity.

A major hurdle to understanding both the organization and the function of microtubules in differentiated cells is a lack of suitable genetic models. Therefore, tools are needed to both visualize and perturb microtubules in vivo. To this end, I generated several novel transgenic mouse lines that allow visualization and perturbation of microtubules in both a spatially and temporally controlled manner in vivo in mice. I generated a TRE-EB1 transgenic mouse line to track growing microtubule plus ends and used this line to image microtubule dynamics in vivo in differentiated keratinocytes for the first time. I showed that differentiation causes suppression of microtubule dynamics in vivo. To perturb microtubules in vivo, I generated a TRE-spastin transgenic mouse, which I use to overexpress the microtubule-severing protein spastin to disrupt microtubules. Using this mouse line, I demonstrate that microtubules are required for different functions in the stratified epidermis versus the simple intestinal epithelium. Disruption of microtubules in differentiated keratinocytes induced profound tissue architecture changes, with many changes in cell shape, differentiation status, and morphogenesis. In striking contrast, intestinal architecture was normal upon microtubule disruption. Instead, we observed relatively subtle defects in trafficking kinetics in the intestinal epithelium. Taken together, this work highlights the value of probing microtubule function in vivo.

There are fundamental differences in the way that neonatal and adult intestines absorb nutrients. In adults, macromolecules are efficiently broken down into simpler molecular components in the lumen of the small intestine, then absorbed. In contrast, neonates are thought to rely more on bulk intake of nutrients and subsequent degradation in the lysosome. Here, we identify the Maf family transcription factors, MafB and cMaf, as markers of terminally-differentiated intestinal enterocytes throughout life. The expression of these factors is regulated by HNF4a/g, master regulators of the enterocyte cell fate. Loss of Maf factors results in a neonatal-specific failure to thrive and loss of bulk uptake of nutrients. RNA-Seq and CUT&RUN analyses defined an endo-lysosomal program as being downstream of these transcription factors. We demonstrate major transcriptional changes in metabolic pathways, including fatty acid oxidation and increases in peroxisome number in response to loss of Mafs. Additionally, we show that deletion of Blimp1, which represses adult enterocyte genes in the neonatal gut, shows highly overlapping changes in gene expression and similar defects in nutrient uptake. This work defines transcriptional regulators that are necessary for bulk uptake in neonatal enterocytes.

Microtubules often adopt non-centrosomal arrays in differentiated tissues, where they are important for providing structure to the cell and maintaining polarity. Although the formation and organization of centrosomal arrays has been well-characterized, little is known about how microtubules form non-centrosomal arrays.

In the mouse epidermis, centrosomes in differentiated cells lose their microtubule-anchoring ability through the loss of proteins from the centrosome. Instead, microtubules are organized around the cell cortex. The cell-cell adhesion protein desmoplakin is required for this organization. Our model is that desmoplakin recruits microtubule-anchoring proteins like ninein to the desmosome, where they subsequently recruit and organize microtubules.

To test this model, we confirmed that the microtubule-binding proteins Lis1, Ndel1, and CLIP170 are recruited by desmoplakin to the cell cortex. Furthermore, by creating an epidermis-specific conditional Lis1 knockout mouse, I found that Lis1 is required for cortical microtubule organization. Surprisingly, however, Lis1 is also required for desmosome stability. This work reveals essential desmosome-associated components that control cortical microtubule organization and unexpected roles for centrosomal proteins in epidermal function.

Although Lis1 is required for microtubule organization, it is not sufficient. I created a culture-based system to determine what other factors may be required for cortical organization for microtubules. My work reveals that stabilization of the microtubules is sufficient to induce their cortical organization. Functionally, cortical microtubules are important for increasing the mechanical integrity of cell sheets by engaging adherens junctions. In turn, tight junction activity is increased. Therefore, I propose that cortical microtubules in the epidermis are important in forming a robust barrier by cooperatively strengthening each cell-cell junction.

To determine whether desmosomes play similar roles in simple epithelia as stratified epithelia, I examined intestinal epithelial-specific conditional desmoplakin conditional knockout mice. Unexpectedly, I found that desmoplakin is not required for cell-cell adhesion and tissue integrity in the small intestine. Furthermore, it does not organize intermediate filaments. Desmoplakin is required, however, for proper microvillus architecture.

Overall, my studies highlight novel tissue-specific roles for desmosomes, in particular desmoplakin, in organizing and integrating different cytoskeletal networks. How desmoplakin's function is regulated in each tissue will be a new interesting area of research.

During 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.

Robust 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.