Self-organization of cell shape and movement

This thesis presents work toward understanding the spatial organization of key molecules during cell morphogenesis and migration. Cell migration is essential for many processes including developmental morphogenesis, axon guidance, and immune responses. Chemotaxis, or directed migration guided by chemical cues, requires the spatial and temporal coordination of a multitude of molecules that pattern the force-generating actin cytoskeleton to build plasma membrane protrusions and power cell motility. This work focuses on identifying novel chemotaxis effectors, dissecting their molecular signaling logic, and exploring how key molecules spatially organize to enable the large-scale, self-organization of cell shape and movement. In the first project, we identified and characterized a novel signaling effector of neutrophil chemotaxis (Chapter 2). From a mass spectrometry pulldown screen, we identified Homer3 as a Gαi2 interacting protein. With biochemical and cell biology techniques, we report that Homer3 is necessary for efficient chemotaxis by regulating the polarized spatial organization, rather than the magnitude and kinetics, of key signaling molecules. Overall, our work characterized how Homer3 functions as a scaffold to spatially organize polarity signaling and actin assembly.In the second project, we studied the spatial organization of the WAVE complex, which is a key effector of cell shape and migration across eukaryotes (Chapter 3). Using quantitative, live-cell super-resolution microscopy, we discovered how the WAVE complex spatially assembles into nanometer scale ring structures at sites of saddle membrane curvature in the absence of actin polymerization. This geometric association for the WAVE complex could explain emergent cell behaviors, such as expanding and self-straightening lamellipodia as well as the ability of endothelial cells to recognize and seal transcellular holes. In the third project, I describe my pilot work using nanotopography to physically manipulate cell geometry to assay curvature sensation (Chapter 4). The interdisciplinary nature of this experiment, which spans nano-engineering, cell biology, and high-resolution microscopy, highlights a combination of expertise that will undoubtedly unveil exciting insights

Clathrin Assembly Defines the Onset and Geometry of Cortical Patterning.

Assembly of the endocytic machinery is a constitutively active process that is important for the organization of the plasma membrane, signal transduction, and membrane trafficking. Existing research has focused on the stochastic nature of endocytosis. Here, we report the emergence of the collective dynamics of endocytic proteins as periodic traveling waves on the cell surface. Coordinated clathrin assembly provides the earliest spatial cue for cortical waves and sets the direction of propagation. Surprisingly, the onset of clathrin waves, but not individual endocytic events, requires feedback from downstream factors, including FBP17, Cdc42, and N-WASP. In addition to the localized endocytic assembly at the plasma membrane, intracellular clathrin and phosphatidylinositol-3,4-bisphosphate predict the excitability of the plasma membrane and modulate the geometry of traveling waves. Collectively, our data demonstrate the multiplicity of clathrin functions in cortical pattern formation and provide important insights regarding the nucleation and propagation of single-cell patterns

When transcription goes on Holliday: Double Holliday junctions block RNA polymerase II transcription in vitro

Non-canonical DNA structures can obstruct transcription. This transcription blockage could have various biological consequences, including genomic instability and gratuitous transcription-coupled repair. Among potential structures causing transcription blockage are Holliday junctions (HJ), which can be generated as intermediates in homologous recombination or during processing of stalled replication forks. Of particular interest is the double Holliday junction (DHJ), which contains two HJs. Topological considerations impose the constraint that the total number of helical turns in the DNA duplexes between the junctions cannot be altered as long as the flanking DNA duplexes are intact. Thus, the DHJ structure should strongly resist transient unwinding during transcription; consequently, it is predicted to cause significantly stronger blockage than single HJ structures. The patterns of transcription blockage obtained for RNA polymerase II transcription in HeLa cell nuclear extracts were in accordance with this prediction. However, we did not detect transcription blockage with purified T7 phage RNA polymerase; we discuss a possible explanation for this difference. In general, our findings implicate naturally occurring Holliday junctions in transcription arrest

Clathrin Assembly Defines the Onset and Geometry of Cortical Patterning.

Assembly of the endocytic machinery is a constitutively active process that is important for the organization of the plasma membrane, signal transduction, and membrane trafficking. Existing research has focused on the stochastic nature of endocytosis. Here, we report the emergence of the collective dynamics of endocytic proteins as periodic traveling waves on the cell surface. Coordinated clathrin assembly provides the earliest spatial cue for cortical waves and sets the direction of propagation. Surprisingly, the onset of clathrin waves, but not individual endocytic events, requires feedback from downstream factors, including FBP17, Cdc42, and N-WASP. In addition to the localized endocytic assembly at the plasma membrane, intracellular clathrin and phosphatidylinositol-3,4-bisphosphate predict the excitability of the plasma membrane and modulate the geometry of traveling waves. Collectively, our data demonstrate the multiplicity of clathrin functions in cortical pattern formation and provide important insights regarding the nucleation and propagation of single-cell patterns

A module for Rac temporal signal integration revealed with optogenetics.

Sensory systems use adaptation to measure changes in signaling inputs rather than absolute levels of signaling inputs. Adaptation enables eukaryotic cells to directionally migrate over a large dynamic range of chemoattractant. Because of complex feedback interactions and redundancy, it has been difficult to define the portion or portions of eukaryotic chemotactic signaling networks that generate adaptation and identify the regulators of this process. In this study, we use a combination of optogenetic intracellular inputs, CRISPR-based knockouts, and pharmacological perturbations to probe the basis of neutrophil adaptation. We find that persistent, optogenetically driven phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production results in only transient activation of Rac, a hallmark feature of adaptive circuits. We further identify the guanine nucleotide exchange factor P-Rex1 as the primary PIP3-stimulated Rac activator, whereas actin polymerization and the GTPase-activating protein ArhGAP15 are essential for proper Rac turnoff. This circuit is masked by feedback and redundancy when chemoattractant is used as the input, highlighting the value of probing signaling networks at intermediate nodes to deconvolve complex signaling cascades

A module for Rac temporal signal integration revealed with optogenetics.

Sensory systems use adaptation to measure changes in signaling inputs rather than absolute levels of signaling inputs. Adaptation enables eukaryotic cells to directionally migrate over a large dynamic range of chemoattractant. Because of complex feedback interactions and redundancy, it has been difficult to define the portion or portions of eukaryotic chemotactic signaling networks that generate adaptation and identify the regulators of this process. In this study, we use a combination of optogenetic intracellular inputs, CRISPR-based knockouts, and pharmacological perturbations to probe the basis of neutrophil adaptation. We find that persistent, optogenetically driven phosphatidylinositol (3,4,5)-trisphosphate (PIP3) production results in only transient activation of Rac, a hallmark feature of adaptive circuits. We further identify the guanine nucleotide exchange factor P-Rex1 as the primary PIP3-stimulated Rac activator, whereas actin polymerization and the GTPase-activating protein ArhGAP15 are essential for proper Rac turnoff. This circuit is masked by feedback and redundancy when chemoattractant is used as the input, highlighting the value of probing signaling networks at intermediate nodes to deconvolve complex signaling cascades

Abstract B48: Homer3 regulates the establishment of neutrophil polarity

Abstract Neutrophils migrate towards developing tumors where they promote angiogenesis, setting the stage for metastasis. Investigating neutrophil migration should lead to novel drug targets to prevent neutrophil accumulation in tumors. Neutrophils and other cells rely on activation of the heterotrimeric G-protein Gai to regulate directional cell migration, but few links from Gai to chemotactic effectors are known. Through affinity chromatography using primary neutrophil lysate, we identify Homer3 as a novel Gai2-binding protein. RNAi-mediated knockdown of Homer3 in neutrophil-like HL-60 cells impairs chemotaxis and the establishment of polarity of the actin cytoskeleton. Most previously-characterized proteins that are required for cell polarity are needed for actin assembly or activation of core chemotactic effectors such as the Rac GTPase. In contrast, Homer3 knockdown cells show a normal magnitude and kinetics of chemoattractant-induced activation of PI3K and Rac effectors. Chemoattractant-stimulated Homer3 knockdown cells also exhibit a normal initial magnitude of actin polymerization, but they fail to polarize actin assembly and are defective in the initiation of cell polarity and motility. Our data suggest that Homer3 acts as a scaffold that spatially organizes actin assembly to support neutrophil polarity and motility downstream of GPCR activation. Citation Format: Julie Wu, Anne Pipathsouk, A Keizer-Gunnink, Wynand Alkema, Fabrizia Fusetti, Shanshan Liu, Steve Atschuler, Lani Wu, Arjan Kortholt, Orion Weiner. Homer3 regulates the establishment of neutrophil polarity. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B48.</jats:p

WAVE complex self-organization templates lamellipodial formation

ABSTRACTHow local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. For example, why does the WAVE complex build lamellipodia, the broad sheet-like protrusions that power cell migration, whereas the homologous actin regulator N-WASP forms spiky finger-like actin networks? N-WASP is known to oligomerize into focal condensates that generate an actin finger. In contrast, the WAVE complex exhibits the linear distribution needed to generate an actin sheet. This linear organization of the WAVE complex could either arise from interactions with the actin cytoskeleton or could represent an ability of the complex to self-organize into a linear template. Using super-resolution microscopy, we find that the WAVE complex forms higher-order linear oligomers that curve into 270 nanometer-wide ring structures in the absence of actin polymer. These rings localize to the necks of membrane invaginations, which display saddle point geometries with positive curvature in one axis and negative curvature in the orthogonal axis. To investigate the molecular mechanism of saddle curvature enrichment, we show that the WAVE complex and IRSp53, a membrane curvature-sensitive protein, collaborate to recognize saddle curvature that IRSp53 cannot sense alone. This saddle preference for the WAVE complex could explain emergent cell behaviors, such as expanding and self-straightening lamellipodia as well as the ability of endothelial cells to recognize and seal transcellular holes. Our work highlights how partnering protein interactions enable complex shape sensing and how feedback between cell shape and actin regulators yields self-organized cell morphogenesis.</jats:p

The WAVE complex associates with sites of saddle membrane curvature.

How local interactions of actin regulators yield large-scale organization of cell shape and movement is not well understood. Here we investigate how the WAVE complex organizes sheet-like lamellipodia. Using super-resolution microscopy, we find that the WAVE complex forms actin-independent 230-nm-wide rings that localize to regions of saddle membrane curvature. This pattern of enrichment could explain several emergent cell behaviors, such as expanding and self-straightening lamellipodia and the ability of endothelial cells to recognize and seal transcellular holes. The WAVE complex recruits IRSp53 to sites of saddle curvature but does not depend on IRSp53 for its own localization. Although the WAVE complex stimulates actin nucleation via the Arp2/3 complex, sheet-like protrusions are still observed in ARP2-null, but not WAVE complex-null, cells. Therefore, the WAVE complex has additional roles in cell morphogenesis beyond Arp2/3 complex activation. Our work defines organizing principles of the WAVE complex lamellipodial template and suggests how feedback between cell shape and actin regulators instructs cell morphogenesis