Cell Biology 2017

The Mexican axolotl (Ambystoma mexicanum) is an aquatic salamander that possesses some of the most astonishing regenerative abilities found in nature.  Able to fully regenerate amputated limbs, the axolotl may hold the key to unlocking similar abilities in others animals.  Salamander limb regeneration has been a source of scientific study and fascination for centuries, but only recently have the cellular and molecular mechanisms behind this process begun to come to light.  Our lab focuses on elucidating the molecular relationship between peripheral nerves, which are essential for regeneration, and the proliferative regenerating limb.  Nerve dependent wound healing and regeneration have been observed across a vast array of animals, and we have found that a certain nerve-derived protein, Neuregulin-1, is essential to this process in axololts and is capable of rescuing limb regeneration even in the absence of nerves.  The goal of this talk is thus to introduce the axolotl as a model for the study of cell and regenerative biology while providing an overview of our recent findings on the molecular basis of nerve dependent tissue growth.

I will present my lab’s effort on studying and manipulating RNA processing, with particular emphasis on using CRISPR/Cas systems for targeting RNA in living cells in diseases such as myotonic dystrophy.

Contractile non-muscle cells, including smooth muscle and myoepithelial cells, provide the mechanical forces required for tissue homeostasis in numerous organ systems. For example, smooth muscle cells surrounding the vasculature contract in response to increased intraluminal pressure to maintain lumen diameter and blood pressure and myoepithelial cells in the mammary gland provide the contractile force required for milk ejection and to shape the mammary gland mechanical microenvironment. Dysregulation of mechano-perception and force production in these contractile cells contributes to the pathophysiology of numerous diseases ranging from hypertension to breast cancer progression. This highlights the importance of understanding how contractility is regulated and maintained in these mechanically stressed cells throughout animal lifetime. To study this question, we use a component of the C. elegans reproductive system, the spermatheca, as a novel model contractile tube. The spermatheca is composed of 24 myoepithelial cells that encompass the sperm. During ovulation, spermathecal cells are dramatically stretched by the incoming oocyte. The oocyte is immediately fertilized and resides in the spermatheca for a regulated period until coordinated spermathecal cell contractions push the embryo into the uterus. This process occurs 150 times throughout the lifetime of the animal requiring robust regulation of tissue contraction to expel the embryo at the correct time and in the correct direction. In this work, we use live animal imaging to monitor Ca2+ signaling and actomyosin dynamics in spermathecal cells during ovulation. We show that Ca2+ transients are produced only after cell stretch and that Ca2+ signaling is required to initiate actomyosin contraction. We further characterize the role of myosin activity in actomyosin network organization and show that both increased and decreased myosin activity alter network properties including connectivity, orientation, and anisotropy. We conclude that tight spatiotemporal regulation of myosin activity throughout successive rounds of ovulation shapes actomyosin network development and maintenance required for tissue function and animal fertility. 

Stem cells can self-renew and differentiate into multiple different cell types. Engineering of stem cells has enabled new methods to study development and organogenesis in humans as well as develop new disease models and therapies. Stem cells can be obtained from multiple sources in vivo or generated in the lab. Engineering of pluripotent stem cells, for example, can potentially generate any tissue in the body and can be used for tissue repair or organoid formation. Furthermore, advances in gene editing technologies have enabled precise editing of stem cell genomes, creating possibilities for establishment of models for diseases and personalized therapies. In this lecture I will discuss the sources and properties of various stem cells as well as their utilization in modelling, therapy and tissue engineering. 

How are your RNA yields? Some sample types present special challenges in RNA purification and analysis. In this webinar, we will discuss and provide tips for the following topics: 

• The basic methods and challenges of RNA purification 
• Special considerations for challenging sample types

Join us to learn how you can apply these practical tips to high quality and quantity of RNA. 

The Resource Identification Initiative supports NIH's new guidelines for Rigor and Transparency as these apply in biomedical publications. Authors are instructed to authenticate key biological resources: Antibodies, Model Organisms, and Tools (software, databases, services), by finding or generating stable unique identifiers. Like a library catalog, these identifiers stick around no matter if the company continues to sell the product or not. This persistence is required for good scholarship and forms a bridge between the static world of the scientific paper and the dynamic world of products and tools.

A fundamental question in neuronal development is how growth cone cytoskeletal dynamics are coordinated to promote accurate axonal navigation. To address this question, we focus on microtubule plus-end tracking proteins (+TIPs), which may play a key role in axon guidance. We determined that TACC3 is a +TIP that promotes microtubule polymerization and axon outgrowth. We have begun to test the hypothesis that TACC3 spatially restricts microtubule polymerization in response to guidance signals in Xenopus laevis axons in vivo and ex vivo. We demonstrate that TACC3 is required to promote axon outgrowth and prevent spontaneous retractions. Additionally, we find that manipulation of TACC3 levels interferes with the growth cone response to axon guidance cues. Finally, we observe that ablation of TACC3 causes pathfinding defects in Xenopus laevis embryos. Together, our findings suggest that TACC3 functions as an axon guidance-regulating factor in embryonic neurons by spatially promoting microtubule polymerization dynamics.

Actin cytoskeleton drives cell migration in muscle and non-muscle cells and plays a major role in such diverse processes as tissue morphogenesis, muscle contraction, and cancer metastases. In non-muscle cells, two major actin isoforms, beta and gamma actin, incorporate into different cytoskeletal structures and mediate cell migration and contractility. In our work we identified that beta, but not gamma actin, is possttranslationally arginylated, and that this arginylation is essential for normal cell migration. Differential arginylation of actin isoforms is regulated at the level of their nucleotide coding sequence. This regulation is a dynamic event that dynamically responds to cellular stimuli and is universal to multiple cell types. Thus, actin arginylation at the cell leading edge is a new regulatory process that acts at post-transcriptional and post-translational levels and is highly selective in targeting actin during cell migration.

The Mexican axolotl (Ambystoma mexicanum) is an aquatic salamander that possesses some of the most astonishing regenerative abilities found in nature.  Able to fully regenerate amputated limbs, the axolotl may hold the key to unlocking similar abilities in others animals.  Salamander limb regeneration has been a source of scientific study and fascination for centuries, but only recently have the cellular and molecular mechanisms behind this process begun to come to light.  Our lab focuses on elucidating the molecular relationship between peripheral nerves, which are essential for regeneration, and the proliferative regenerating limb.  Nerve dependent wound healing and regeneration have been observed across a vast array of animals, and we have found that a certain nerve-derived protein, Neuregulin-1, is essential to this process in axololts and is capable of rescuing limb regeneration even in the absence of nerves.  The goal of this talk is thus to introduce the axolotl as a model for the study of cell and regenerative biology while providing an overview of our recent findings on the molecular basis of nerve dependent tissue growth.

Einstein researcher Robert Singer, Ph.D., discusses a breakthrough in microscopy that is allowing scientists to track messenger RNA in living cells in real time. The study, published in the September 15 online edition of Nature, marks a major advance in the use of microscopes for scientific investigation and the findings could lead to treatments for a variety of disorders, such as myotonic dystrophy, in which mRNA gets stuck inside the nucleus of cells.

The manipulation of the genome to study disease, evaluate drug targets and to stratify patient populations is now a widely accepted concept in the field. At Horizon Discovery, genome engineering is the core of everything we do and following the breakthrough in 2013, with numerous publications into the capabilities of CRISPR-Cas9 engineering, the use and applications of this technology have grown exponentially. Our selection of cell lines and engineering capabilities allows you to bring definition to your research with genotype specific cell line models.
In order to be successful with CRISPR-Cas9 genome engineering, there are key considerations that need to be addressed, from the cell line you want to use, to guide design and screening for the mutations of interest. In this presentation, we will discuss each stage of the experimental design and will introduce you to several cutting-edge applications of CRISPR-Cas9 including generation of reporter cell lines and the development of new variations on this technology, CRISPRa and CRISPRi.

Although the cell is commonly referred to as “the most basic unit of life”, it is actually so complex that despite over 350 years of research we are still far from fully understanding its structural, functional and evolutionary workings. Even the simplest unicellular organisms routinely carry out complicated tasks such as processing and responding to signals from the environment, maintaining an efficient metabolic network, communicating with other cells, and adapting to new evolutionary challenges.

Studies in cell biology have illuminated many molecular pathways and protein functions that govern cells, but we still do not fully understand how these complex systems are established and function. I, and others, argue that a better dissection of the evolutionary mechanisms that produce cellular functions will significantly advance our understanding of the fundamental principles governing the complexities of cell biological systems by providing the rationale behind variations in cellular functions and networks. Bringing the fields of cell biology and evolution together into an integrated field of “evolutionary cell biology” has historically produced major advances in our understanding of cells. For example, the development of the “endosymbiotic theory” of how mitochondria arose revolutionized our understanding of the origins and structure of eukaryotic cells. Hence, further development of this interdisciplinary field will ultimately allow us to better address diverse areas of cell biology.

Throughout my PhD, I found that combining evolutionary and cell-biological studies brings unique insights on cellular innovation, complexity, limitation, and adaptation. To this end, in my research, I combined molecular genetics methods and large-scale synthetic libraries with long-term lab-evolution protocols. With this approach, I manipulated the translation and splicing machineries, separately, to present them with molecular challenges. I then followed and deciphered the adaptation mechanisms used by these complex systems to restore their proper function. My work demonstrates how an evolutionary perspective on cell biological questions can teach us about the workings of living cells.

How are your RNA yields? Some sample types present special challenges in RNA purification and analysis. In this webinar, we will discuss and provide tips for the following topics: 

• The basic methods and challenges of RNA purification 
• Special considerations for challenging sample types

Join us to learn how you can apply these practical tips to high quality and quantity of RNA. 

To study cell metabolism, our laboratory has found it exceedingly informative to integrate Agilent Seahorse XF Analyzers with mass spectrometry-based measurements of metabolomics and stable isotope tracing to fully reveal metabolic changes in response to drug candidates.  This seminar will discuss how merging quantitative, rate-based measurements of oxygen consumption with detailed pathway analysis using mass spectrometry have revealed the mitochondrial pyruvate carrier (MPC) as a central regulator of metabolic flexibility. Metabolic flexibility is the ability of cells to fuel energy metabolism and biosynthesis with multiple nutrients, and its loss is associated with disease pathogenesis in contexts as varied as type 2 diabetes, cardiac dysfunction, neurodegeneration, and certain cancers. 

For Research Use Only. Not for use in diagnostic procedures. 

Checkpoint blockade therapy for cancer has had tremendous impact on clinical outcomes, yet only a subset of patients respond. Recent studies show that response to checkpoint blockade does not always correlate with tumor-associated antigen (TAA) load and so must be determined by factors beyond mutational burden. This suggests that checkpoint blockade is limited by suboptimal cross-presentation of TAA by activated dendritic cells (DC) and will be potentiated by recruitment, loading and activation of cross-presenting DC at the tumor site.

To test this hypothesis, an early-phase trial (NCT01976585) in low-grade lymphoma was carried out testing a unique in situ vaccine (ISV) combining: 1) fms-like tyrosine kinase 3 ligand (FLT3L) to recruit DC, 2) radiotherapy to load FLT3L-mobilized DC with TAA, and 3) toll-like receptor agonist (TLRa) to activate TAA-loaded DC for cross-presentation. Strikingly, Brody and colleagues found partial and complete systemic tumor regressions at distant, untreated tumors. They also found specific elimination of malignant B cells with sparing of healthy B cells, suggesting a systemic anti-tumor immune response.

This data prompted a new trial in which anti-PD1 monoclonal antibody (mAb) and ISV (used to increase efficacy) are combined with a novel immune-monitoring approach involving co-administration of "surrogate antigens (Ag)" at the ISV site. The approach is being applied to multiple cancer types, including breast, head and neck, melanoma and sarcoma.

In this webinar, Brody will discuss these studies, including the key role mass cytometry played in defining the effects of treatment on the intratumoral and systemic immune repertoire with high resolution and in profiling the distinct checkpoint/co-activator molecules on TAA-specific, surrogate-Ag-specific and bulk CD8 T cells.

Contractile non-muscle cells, including smooth muscle and myoepithelial cells, provide the mechanical forces required for tissue homeostasis in numerous organ systems. For example, smooth muscle cells surrounding the vasculature contract in response to increased intraluminal pressure to maintain lumen diameter and blood pressure and myoepithelial cells in the mammary gland provide the contractile force required for milk ejection and to shape the mammary gland mechanical microenvironment. Dysregulation of mechano-perception and force production in these contractile cells contributes to the pathophysiology of numerous diseases ranging from hypertension to breast cancer progression. This highlights the importance of understanding how contractility is regulated and maintained in these mechanically stressed cells throughout animal lifetime. To study this question, we use a component of the C. elegans reproductive system, the spermatheca, as a novel model contractile tube. The spermatheca is composed of 24 myoepithelial cells that encompass the sperm. During ovulation, spermathecal cells are dramatically stretched by the incoming oocyte. The oocyte is immediately fertilized and resides in the spermatheca for a regulated period until coordinated spermathecal cell contractions push the embryo into the uterus. This process occurs 150 times throughout the lifetime of the animal requiring robust regulation of tissue contraction to expel the embryo at the correct time and in the correct direction. In this work, we use live animal imaging to monitor Ca2+ signaling and actomyosin dynamics in spermathecal cells during ovulation. We show that Ca2+ transients are produced only after cell stretch and that Ca2+ signaling is required to initiate actomyosin contraction. We further characterize the role of myosin activity in actomyosin network organization and show that both increased and decreased myosin activity alter network properties including connectivity, orientation, and anisotropy. We conclude that tight spatiotemporal regulation of myosin activity throughout successive rounds of ovulation shapes actomyosin network development and maintenance required for tissue function and animal fertility. 

I will present my lab’s effort on studying and manipulating RNA processing, with particular emphasis on using CRISPR/Cas systems for targeting RNA in living cells in diseases such as myotonic dystrophy.

The Resource Identification Initiative supports NIH's new guidelines for Rigor and Transparency as these apply in biomedical publications. Authors are instructed to authenticate key biological resources: Antibodies, Model Organisms, and Tools (software, databases, services), by finding or generating stable unique identifiers. Like a library catalog, these identifiers stick around no matter if the company continues to sell the product or not. This persistence is required for good scholarship and forms a bridge between the static world of the scientific paper and the dynamic world of products and tools.

Stem cells can self-renew and differentiate into multiple different cell types. Engineering of stem cells has enabled new methods to study development and organogenesis in humans as well as develop new disease models and therapies. Stem cells can be obtained from multiple sources in vivo or generated in the lab. Engineering of pluripotent stem cells, for example, can potentially generate any tissue in the body and can be used for tissue repair or organoid formation. Furthermore, advances in gene editing technologies have enabled precise editing of stem cell genomes, creating possibilities for establishment of models for diseases and personalized therapies. In this lecture I will discuss the sources and properties of various stem cells as well as their utilization in modelling, therapy and tissue engineering.