Prolonged depletion of profilin 1 or F-actin causes an adaptive response in microtubules.

In addition to its well-established role in actin assembly, profilin 1 (PFN1) has been shown to bind to tubulin and alter microtubule growth. However, whether PFN1's predominant control over microtubules in cells occurs through direct regulation of tubulin or indirectly through the polymerization of actin has yet to be determined. Here, we manipulated PFN1 expression, actin filament assembly, and actomyosin contractility and showed that reducing any of these parameters for extended periods of time caused an adaptive response in the microtubule cytoskeleton, with the effect being significantly more pronounced in neuronal processes. All the observed changes to microtubules were reversible if actomyosin was restored, arguing that PFN1's regulation of microtubules occurs principally through actin. Moreover, the cytoskeletal modifications resulting from PFN1 depletion in neuronal processes affected microtubule-based transport and mimicked phenotypes that are linked to neurodegenerative disease. This demonstrates how defects in actin can cause compensatory responses in other cytoskeleton components, which in turn significantly alter cellular function.

The actin binding protein profilin 1 is critical for mitochondria function.

Profilin 1 (PFN1) is an actin binding protein that is vital for the polymerization of monomeric actin into filaments. Here we screened knockout cells for novel functions of PFN1 and discovered that mitophagy, a type of selective autophagy that removes defective or damaged mitochondria from the cell, was significantly upregulated in the absence of PFN1. Despite successful autophagosome formation and fusion with the lysosome, and activation of additional mitochondrial quality control pathways, PFN1 knockout cells still accumulate damaged, dysfunctional mitochondria. Subsequent imaging and functional assays showed that loss of PFN1 significantly affects mitochondria morphology, dynamics, and respiration. Further experiments revealed that PFN1 is located to the mitochondria matrix and is likely regulating mitochondria function from within rather than through polymerizing actin at the mitochondria surface. Finally, PFN1 mutants associated with amyotrophic lateral sclerosis (ALS) fail to rescue PFN1 knockout mitochondrial phenotypes and form aggregates within mitochondria, further perturbing them. Together, these results suggest a novel function for PFN1 in regulating mitochondria and identify a potential pathogenic mechanism of ALS-linked PFN1 variants.

Protein disulfide isomerase A1 as a novel redox sensor in VEGFR2 signaling and angiogenesis.

VEGFR2 signaling in endothelial cells (ECs) is regulated by reactive oxygen species (ROS) derived from NADPH oxidases (NOXs) and mitochondria, which plays an important role in postnatal angiogenesis. However, it remains unclear how highly diffusible ROS signal enhances VEGFR2 signaling and reparative angiogenesis. Protein disulfide isomerase A1 (PDIA1) functions as an oxidoreductase depending on the redox environment. We hypothesized that PDIA1 functions as a redox sensor to enhance angiogenesis. Here we showed that PDIA1 co-immunoprecipitated with VEGFR2 or colocalized with either VEGFR2 or an early endosome marker Rab5 at the perinuclear region upon stimulation of human ECs with VEGF. PDIA1 silencing significantly reduced VEGF-induced EC migration, proliferation and spheroid sprouting via inhibiting VEGFR2 signaling. Mechanistically, VEGF stimulation rapidly increased Cys-OH formation of PDIA1 via the NOX4-mitochondrial ROS axis. Overexpression of "redox-dead" mutant PDIA1 with replacement of the active four Cys residues with Ser significantly inhibited VEGF-induced PDIA1-CysOH formation and angiogenic responses via reducing VEGFR2 phosphorylation. Pdia1+/- mice showed impaired angiogenesis in developmental retina and Matrigel plug models as well as ex vivo aortic ring sprouting model. Study using hindlimb ischemia model revealed that PDIA1 expression was markedly increased in angiogenic ECs of ischemic muscles, and that ischemia-induced limb perfusion recovery and neovascularization were impaired in EC-specific Pdia1 conditional knockout mice. These results suggest that PDIA1 can sense VEGF-induced H2O2 signal via CysOH formation to promote VEGFR2 signaling and angiogenesis in ECs, thereby enhancing postnatal angiogenesis. The oxidized PDIA1 is a potential therapeutic target for treatment of ischemic vascular diseases.

TDAExplore: Quantitative analysis of fluorescence microscopy images through topology-based machine learning.

Recent advances in machine learning have greatly enhanced automatic methods to extract information from fluorescence microscopy data. However, current machine-learning-based models can require hundreds to thousands of images to train, and the most readily accessible models classify images without describing which parts of an image contributed to classification. Here, we introduce TDAExplore, a machine learning image analysis pipeline based on topological data analysis. It can classify different types of cellular perturbations after training with only 20-30 high-resolution images and performs robustly on images from multiple subjects and microscopy modes. Using only images and whole-image labels for training, TDAExplore provides quantitative, spatial information, characterizing which image regions contribute to classification. Computational requirements to train TDAExplore models are modest and a standard PC can perform training with minimal user input. TDAExplore is therefore an accessible, powerful option for obtaining quantitative information about imaging data in a wide variety of applications.

Delivering defined amounts of purified protein with high precision into living cells.

Here, we detail a protocol using electroporation to precisely deliver defined amounts of purified protein into CAD cells. This method allows one million cells to be electroporated with protein simultaneously, with high delivery efficiency and low cell death. Further, by circumventing the normal biosynthetic pathway, proteins can be studied without the complication of post-translational modifications and before a transcriptional response can be initiated. This protocol will be useful for any researcher who is interested in protein concentration-dependent cellular phenotypes. For complete details on the use and execution of this protocol, please refer to Skruber et al. (2020).

Arp2/3 and Mena/VASP Require Profilin 1 for Actin Network Assembly at the Leading Edge.

Cells have many types of actin structures, which must assemble from a common monomer pool. Yet, it remains poorly understood how monomers are distributed to and shared between different filament networks. Simplified model systems suggest that monomers are limited and heterogeneous, which alters actin network assembly through biased polymerization and internetwork competition. However, less is known about how monomers influence complex actin structures, where different networks competing for monomers overlap and are functionally interdependent. One example is the leading edge of migrating cells, which contains filament networks generated by multiple assembly factors. The leading edge dynamically switches between the formation of different actin structures, such as lamellipodia or filopodia, by altering the balance of these assembly factors' activities. Here, we sought to determine how the monomer-binding protein profilin 1 (PFN1) controls the assembly and organization of actin in mammalian cells. Actin polymerization in PFN1 knockout cells was severely disrupted, particularly at the leading edge, where both Arp2/3 and Mena/VASP-based filament assembly was inhibited. Further studies showed that in the absence of PFN1, Arp2/3 no longer localizes to the leading edge and Mena/VASP is non-functional. Additionally, we discovered that discrete stages of internetwork competition and collaboration between Arp2/3 and Mena/VASP networks exist at different PFN1 concentrations. Low levels of PFN1 caused filopodia to form exclusively at the leading edge, while higher concentrations inhibited filopodia and favored lamellipodia and pre-filopodia bundles. These results demonstrate that dramatic changes to actin architecture can be made simply by modifying PFN1 availability.

ALS-Linked SOD1 Mutants Enhance Neurite Outgrowth and Branching in Adult Motor Neurons.

Amyotrophic lateral sclerosis (ALS) is a progressive, fatal neurodegenerative disease characterized by motor neuron cell death. However, not all motor neurons are equally susceptible. Most of what we know about the surviving motor neurons comes from gene expression profiling; less is known about their functional traits. We found that resistant motor neurons cultured from SOD1 ALS mouse models have enhanced axonal outgrowth and dendritic branching. They also have an increase in the number and size of actin-based structures like growth cones and filopodia. These phenotypes occur in cells cultured from presymptomatic mice and mutant SOD1 models that do not develop ALS but not in embryonic motor neurons. Enhanced outgrowth and upregulation of filopodia can be induced in wild-type adult cells by expressing mutant SOD1. These results demonstrate that mutant SOD1 can enhance the regenerative capability of ALS-resistant motor neurons. Capitalizing on this mechanism could lead to new therapeutic strategies.

Reconsidering an active role for G-actin in cytoskeletal regulation.

Globular (G)-actin, the actin monomer, assembles into polarized filaments that form networks that can provide structural support, generate force and organize the cell. Many of these structures are highly dynamic and to maintain them, the cell relies on a large reserve of monomers. Classically, the G-actin pool has been thought of as homogenous. However, recent work has shown that actin monomers can exist in distinct groups that can be targeted to specific networks, where they drive and modify filament assembly in ways that can have profound effects on cellular behavior. This Review focuses on the potential factors that could create functionally distinct pools of actin monomers in the cell, including differences between the actin isoforms and the regulation of G-actin by monomer binding proteins, such as profilin and thymosin ß4. Owing to difficulties in studying and visualizing G-actin, our knowledge over the precise role that specific actin monomer pools play in regulating cellular actin dynamics remains incomplete. Here, we discuss some of these unanswered questions and also provide a summary of the methodologies currently available for the imaging of G-actin.

Simultaneous quantification of actin monomer and filament dynamics with modeling-assisted analysis of photoactivation.

Photoactivation allows one to pulse-label molecules and obtain quantitative data about their behavior. We have devised a new modeling-based analysis for photoactivatable actin experiments that simultaneously measures properties of monomeric and filamentous actin in a three-dimensional cellular environment. We use this method to determine differences in the dynamic behavior of ß- and γ-actin isoforms, showing that both inhabit filaments that depolymerize at equal rates but that ß-actin exists in a higher monomer-to-filament ratio. We also demonstrate that cofilin (cofilin 1) equally accelerates depolymerization of filaments made from both isoforms, but is only required to maintain the ß-actin monomer pool. Finally, we used modeling-based analysis to assess actin dynamics in axon-like projections of differentiating neuroblastoma cells, showing that the actin monomer concentration is significantly depleted as the axon develops. Importantly, these results would not have been obtained using traditional half-time analysis. Given that parameters of the publicly available modeling platform can be adjusted to suit the experimental system of the user, this method can easily be used to quantify actin dynamics in many different cell types and subcellular compartments.

HDM2 promotes WIP1-mediated medulloblastoma growth.

Medulloblastoma is the most common malignant childhood brain tumor. The protein phosphatase and oncogene WIP1 is over-expressed or amplified in a significant number of primary human medulloblastomas and cell lines. In the present study, we examine an important mechanism by which WIP1 promotes medulloblastoma growth using in vitro and in vivo models. Human cell lines and intracerebellar xenografted animal models were used to study the role of WIP1 and the major TP53 regulator, HDM2, in medulloblastoma growth. Stable expression of WIP1 enhances growth of TP53 wild-type medulloblastoma cells, compared with cells with stable expression of an empty-vector or mutant WIP1. In an animal model, WIP1 enhances proliferation and reduces the survival of immunodeficient mice bearing intracerebellar xenografted human medulloblastoma cells. Cells with increased WIP1 expression also exhibit increased expression of HDM2. HDM2 knockdown or treatment with the HDM2 inhibitor Nutlin-3a, the active enantomer of Nutlin-3, specifically inhibits the growth of medulloblastoma cells with increased WIP1 expression. Nutlin-3a does not affect growth of medulloblastoma cells with stable expression of an empty vector or of mutant WIP1. Knockdown of WIP1 or treatment with the WIP1 inhibitor CCT007093 results in increased phosphorylation of known WIP1 targets, reduced HDM2 expression, and reduced growth specifically in WIP1 wild-type and high-expressing medulloblastoma cells. Combined WIP1 and HDM2 inhibition is more effective than WIP1 inhibition alone in blocking growth of WIP1 high-expressing medulloblastoma cells. Our preclinical study supports a role for therapies that target WIP1 and HDM2 in the treatment of medulloblastoma.

An animal model of MYC-driven medulloblastoma.

Medulloblastoma (MB) is the most common malignant brain tumor in children. Patients whose tumors exhibit overexpression or amplification of the MYC oncogene (c-MYC) usually have an extremely poor prognosis, but there are no animal models of this subtype of the disease. Here, we show that cerebellar stem cells expressing Myc and mutant Trp53 (p53) generate aggressive tumors following orthotopic transplantation. These tumors consist of large, pleiomorphic cells and resemble human MYC-driven MB at a molecular level. Notably, antagonists of PI3K/mTOR signaling, but not Hedgehog signaling, inhibit growth of tumor cells. These findings suggest that cerebellar stem cells can give rise to MYC-driven MB and identify a novel model that can be used to test therapies for this devastating disease.

Spheres without influence: dissociating in vitro self-renewal from tumorigenic potential in glioma.

The capacity for self-renewal is thought to be a critical property of tumor-initiating cells. This capacity is often associated with the ability to generate spheres in vitro. In this issue of Cancer Cell, Barrett et al. show that cells lacking sphere-forming ability can still be very efficient at propagating tumors.

Identification of CD15 as a marker for tumor-propagating cells in a mouse model of medulloblastoma.

The growth of many cancers depends on self-renewing cells called cancer stem cells or tumor-propagating cells (TPCs). In human brain tumors, cells expressing the stem cell marker CD133 have been implicated as TPCs. Here we show that tumors from a model of medulloblastoma, the Patched mutant mouse, are propagated not by CD133(+) cells but by cells expressing the progenitor markers Math1 and CD15/SSEA-1. These cells have a distinct expression profile that suggests increased proliferative capacity and decreased tendency to undergo apoptosis and differentiation. CD15 is also found in a subset of human medulloblastomas, and tumors expressing genes similar to those found in murine CD15(+) cells have a poorer prognosis. Thus, CD15 may represent an important marker for TPCs in medulloblastoma.

Medulloblastoma can be initiated by deletion of Patched in lineage-restricted progenitors or stem cells.

Medulloblastoma is the most common malignant brain tumor in children, but the cells from which it arises remain unclear. Here we examine the origin of medulloblastoma resulting from mutations in the Sonic hedgehog (Shh) pathway. We show that activation of Shh signaling in neuronal progenitors causes medulloblastoma by 3 months of age. Shh pathway activation in stem cells promotes stem cell proliferation but only causes tumors after commitment to-and expansion of-the neuronal lineage. Notably, tumors initiated in stem cells develop more rapidly than those initiated in progenitors, with all animals succumbing by 3-4 weeks. These studies suggest that medulloblastoma can be initiated in progenitors or stem cells but that Shh-induced tumorigenesis is associated with neuronal lineage commitment.

The neurobiology of neurooncology.

The histological classification of brain tumors currently is based on the morphological appearance and protein expression patterns that reflect specific cell types within the central nervous system. Recent studies have suggested that the cells of origin for brain tumors may persist in the fully formed tumors, and that these "cancer stem cells" might represent the relevant cellular targets for anticancer therapy. In this regard, insights into the developmental neurobiology of brain tumors has significant impact on our understanding of the molecular and cellular pathogenesis of these devastating cancers, as well as the development of new strategies for treating brain tumors.

Dominant-negative inhibition of the Axl receptor tyrosine kinase suppresses brain tumor cell growth and invasion and prolongs survival.

Malignant gliomas remain incurable brain tumors because of their diffuse-invasive growth. So far, the genetic and molecular events underlying gliomagenesis are poorly understood. In this study, we have identified the receptor tyrosine kinase Axl as a mediator of glioma growth and invasion. We demonstrate that Axl and its ligand Gas6 are overexpressed in human glioma cell lines and that Axl is activated under baseline conditions. Furthermore, Axl is expressed at high levels in human malignant glioma. Inhibition of Axl signaling by overexpression of a dominant-negative receptor mutant (AXL-DN) suppressed experimental gliomagenesis (growth inhibition >85%, P < 0.05) and resulted in long-term survival of mice after intracerebral glioma cell implantation when compared with Axl wild-type (AXL-WT) transfected tumor cells (survival times: AXL-WT, 10 days; AXL-DN, >72 days). A detailed analysis of the distinct hallmarks of glioma pathology, such as cell proliferation, migration, and invasion and tumor angiogenesis, revealed that inhibition of Axl signaling interfered with cell proliferation (inhibition 30% versus AXL-WT), glioma cell migration (inhibition 90% versus mock and AXL-WT, P < 0.05), and invasion (inhibition 62% and 79% versus mock and AXL-WT, respectively; P < 0.05). This study describes the identification, functional manipulation, in vitro and in vivo validation, and preclinical therapeutic inhibition of a target receptor tyrosine kinase mediating glioma growth and invasion. Our findings implicate Axl in gliomagenesis and validate it as a promising target for the development of approaches toward a therapy of these highly aggressive but, as yet, therapy-refractory, tumors.

Isolation of neural stem cells from the postnatal cerebellum.

The cerebellum is critical for motor coordination and cognitive function and is the target of transformation in medulloblastoma, the most common malignant brain tumor in children. Although the development of granule cells, the most abundant neurons in the cerebellum, has been studied in detail, the origins of other cerebellar neurons and glia remain poorly understood. Here we show that the murine postnatal cerebellum contains multipotent neural stem cells (NSCs). These cells can be prospectively isolated based on their expression of the NSC marker prominin-1 (CD133) and their lack of markers of neuronal and glial lineages (lin-). Purified prominin+ lin- cells form self-renewing neurospheres and can differentiate into astrocytes, oligodendrocytes and neurons in vitro. Moreover, they can generate each of these lineages after transplantation into the cerebellum. Identification of cerebellar stem cells has important implications for the understanding of cerebellar development and the origins of medulloblastoma.

Loss of patched and disruption of granule cell development in a pre-neoplastic stage of medulloblastoma.

Medulloblastoma is the most common malignant brain tumor in children. It is thought to result from the transformation of granule cell precursors (GCPs) in the developing cerebellum, but little is known about the early stages of the disease. Here, we identify a pre-neoplastic stage of medulloblastoma in patched heterozygous mice, a model of the human disease. We show that pre-neoplastic cells are present in the majority of patched mutants, although only 16% of these mice develop tumors. Pre-neoplastic cells, like tumor cells, exhibit activation of the Sonic hedgehog pathway and constitutive proliferation. Importantly, they also lack expression of the wild-type patched allele, suggesting that loss of patched is an early event in tumorigenesis. Although pre-neoplastic cells resemble GCPs and tumor cells in many respects, they have a distinct molecular signature. Genes that mark the pre-neoplastic stage include regulators of migration, apoptosis and differentiation, processes crucial for normal development but previously unrecognized for their role in medulloblastoma. The identification and molecular characterization of pre-neoplastic cells provides insight into the early steps in medulloblastoma formation, and may yield important markers for early detection and therapy of this disease.

Expression of extracellular matrix components in a highly infiltrative in vivo glioma model.

This work demonstrates the expression of extracellular matrix (ECM) components in a highly infiltrative brain tumor model developed by simple inoculation of spheroids from five human glioma biopsy tissues directly into the brains of immunodeficient rats. Non-invasive tumors derived from one glioblastoma biopsy specimen and two glioma cell lines (D-54MG and U-251MG) were also included in this study. The extent of tumor cell infiltration was studied using a pan-human monoclonal anti-vimentin antibody. The cellular origin for several of these ECM components was identified using human-specific monoclonal antibodies and polyclonal antibodies detecting epitopes from both species. Immunostaining revealed a diffuse parenchymal staining of glioma-produced tenascin, whereas vitronectin was produced mainly by the invading glioma cells. ECM components such as laminin, fibronectin and collagen type IV were most probably produced by the host and were mainly associated with the blood vessels in the tumors. However, some parenchymal staining with regional variations was observed. The expression pattern of these components was different in cell lines tumors as compared to the biopsy specimen tumors. The alpha3 and beta1 integrin subunits were mainly observed in areas of tumor cell invasion in the invasive tumors. In conclusion, the observed staining patterns clarify the cellular origin and indicate the possible biological function of tenascin, vitronectin, laminin, fibronectin and collagen type IV in these highly invasive malignant tumors of glial origin.