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.

Cytoskeletal adaptation following long-term dysregulation of actomyosin in neuronal processes.

Microtubules, intermediate filaments, and actin are cytoskeletal polymer networks found within the cell. While each has unique functions, all the cytoskeletal elements must work together for cellular mechanics to be fully operative. This is achieved through crosstalk mechanisms whereby the different networks influence each other through signaling pathways and direct interactions. Because crosstalk can be complex, it is possible for perturbations in one cytoskeletal element to affect the others in ways that are difficult to predict. Here we investigated how long-term changes to the actin cytoskeleton affect microtubules and intermediate filaments. Reducing F-actin or actomyosin contractility increased acetylated microtubules and intermediate filament expression, with the effect being significantly more pronounced in neuronal processes. Changes to microtubules were completely reversible if F-actin and myosin activity is restored. Moreover, the altered microtubules in neuronal processes resulting from F-actin depletion caused significant changes to microtubule-based transport, mimicking phenotypes that are linked to neurodegenerative disease. Thus, defects in actin dynamics cause a compensatory response in other cytoskeleton components which profoundly alters 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.

Nonmuscle myosin 2 filaments are processive in cells.

Directed transport of cellular components is often dependent on the processive movements of cytoskeletal motors. Myosin 2 motors predominantly engage actin filaments of opposing orientation to drive contractile events and are therefore not traditionally viewed as processive. However, recent in vitro experiments with purified nonmuscle myosin 2 (NM2) demonstrated myosin 2 filaments could move processively. Here, we establish processivity as a cellular property of NM2. Processive runs in central nervous system-derived CAD cells are most apparent on bundled actin in protrusions that terminate at the leading edge. We find that processive velocities in vivo are consistent with in vitro measurements. NM2 makes these processive runs in its filamentous form against lamellipodia retrograde flow, though anterograde movement can still occur in the absence of actin dynamics. Comparing the processivity of NM2 isoforms, we find that NM2A moves slightly faster than NM2B. Finally, we demonstrate that this is not a cell-specific property, as we observe processive-like movements of NM2 in the lamella and subnuclear stress fibers of fibroblasts. Collectively, these observations further broaden NM2 functionality and the biological processes in which the already ubiquitous motor can contribute.

Non-muscle myosin 2 filaments are processive in cells.

Directed transport of cellular components is often dependent on the processive movements of cytoskeletal motors. Myosin 2 motors predominantly engage actin filaments of opposing orientation to drive contractile events, and are therefore not traditionally viewed as processive. However, recent in vitro experiments with purified non-muscle myosin 2 (NM2) demonstrated myosin 2 filaments could move processively. Here, we establish processivity as a cellular property of NM2. Processive runs in central nervous system-derived CAD cells are most apparent as processive movements on bundled actin in protrusions that terminate at the leading edge. We find that processive velocities in vivo are consistent with in vitro measurements. NM2 makes these processive runs in its filamentous form against lamellipodia retrograde flow, though anterograde movement can still occur in the absence of actin dynamics. Comparing the processivity of NM2 isoforms, we find that NM2A moves slightly faster than NM2B. Finally, we demonstrate that this is not a cell-specific property, as we observe processive-like movements of NM2 in the lamella and subnuclear stress fibers of fibroblasts. Collectively, these observations further broaden NM2 functionality and the biological processes in which the already ubiquitous motor can contribute.

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.

Cell-type-specific dysregulation of RNA alternative splicing in short tandem repeat mouse knockin models of myotonic dystrophy.

Short tandem repeats (STRs) are prone to expansion mutations that cause multiple hereditary neurological and neuromuscular diseases. To study pathomechanisms using mouse models that recapitulate the tissue specificity and developmental timing of an STR expansion gene, we used rolling circle amplification and CRISPR/Cas9-mediated genome editing to generate Dmpk CTG expansion (CTGexp) knockin models of myotonic dystrophy type 1 (DM1). We demonstrate that skeletal muscle myoblasts and brain choroid plexus epithelial cells are particularly susceptible to Dmpk CTGexp mutations and RNA missplicing. Our results implicate dysregulation of muscle regeneration and cerebrospinal fluid homeostasis as early pathogenic events in DM1.

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.

Two functionally distinct sources of actin monomers supply the leading edge of lamellipodia.

Lamellipodia, the sheet-like protrusions of motile cells, consist of networks of actin filaments (F-actin) regulated by the ordered assembly from and disassembly into actin monomers (G-actin). Traditionally, G-actin is thought to exist as a homogeneous pool. Here, we show that there are two functionally and molecularly distinct sources of G-actin that supply lamellipodial actin networks. G-actin originating from the cytosolic pool requires the monomer-binding protein thymosin ß4 (Tß4) for optimal leading-edge localization, is targeted to formins, and is responsible for creating an elevated G/F-actin ratio that promotes membrane protrusion. The second source of G-actin comes from recycled lamellipodia F-actin. Recycling occurs independently of Tß4 and appears to regulate lamellipodia homeostasis. Tß4-bound G-actin specifically localizes to the leading edge because it does not interact with Arp2/3-mediated polymerization sites found throughout the lamellipodia. These findings demonstrate that actin networks can be constructed from multiple sources of monomers with discrete spatiotemporal functions.

CellGeo: a computational platform for the analysis of shape changes in cells with complex geometries.

Cell biologists increasingly rely on computer-aided image analysis, allowing them to collect precise, unbiased quantitative results. However, despite great progress in image processing and computer vision, current computational approaches fail to address many key aspects of cell behavior, including the cell protrusions that guide cell migration and drive morphogenesis. We developed the open source MATLAB application CellGeo, a user-friendly computational platform to allow simultaneous, automated tracking and analysis of dynamic changes in cell shape, including protrusions ranging from filopodia to lamellipodia. Our method maps an arbitrary cell shape onto a tree graph that, unlike traditional skeletonization algorithms, preserves complex boundary features. CellGeo allows rigorous but flexible definition and accurate automated detection and tracking of geometric features of interest. We demonstrate CellGeo's utility by deriving new insights into (a) the roles of Diaphanous, Enabled, and Capping protein in regulating filopodia and lamellipodia dynamics in Drosophila melanogaster cells and (b) the dynamic properties of growth cones in catecholaminergic a-differentiated neuroblastoma cells.

Dynamic localization of G-actin during membrane protrusion in neuronal motility.

BACKGROUND: Actin-based cell motility is fundamental for development, function, and malignant events in eukaryotic organisms. During neural development, axonal growth cones depend on rapid assembly and disassembly of actin filaments (F-actin) for their guided extension to specific targets for wiring. Monomeric globular actin (G-actin) is the building block for F-actin but is not considered to play a direct role in spatiotemporal control of actin dynamics in cell motility. RESULTS: Here we report that a pool of G-actin dynamically localizes to the leading edge of growth cones and neuroblastoma cells to spatially elevate the G-/F-actin ratio that drives membrane protrusion and cell movement. Loss of G-actin localization leads to the cessation and retraction of membrane protrusions. Moreover, G-actin localization occurs asymmetrically in growth cones during attractive turning. Finally, we identify the actin monomer-binding proteins profilin and thymosin ß4 as key molecules that localize actin monomers to the leading edge of lamellipodia for their motility. CONCLUSIONS: Our results suggest that dynamic localization of G-actin provides a novel mechanism to regulate the spatiotemporal actin dynamics underlying membrane protrusion in cell locomotion and growth cone chemotaxis.

Instantaneous inactivation of cofilin reveals its function of F-actin disassembly in lamellipodia.

Cofilin is a key regulator of the actin cytoskeleton. It can sever actin filaments, accelerate filament disassembly, act as a nucleation factor, recruit or antagonize other actin regulators, and control the pool of polymerization-competent actin monomers. In cells these actions have complex functional outputs. The timing and localization of cofilin activity are carefully regulated, and thus global, long-term perturbations may not be sufficient to probe its precise function. To better understand cofilin's spatiotemporal action in cells, we implemented chromophore-assisted laser inactivation (CALI) to instantly and specifically inactivate it. In addition to globally inhibiting actin turnover, CALI of cofilin generated several profound effects on the lamellipodia, including an increase of F-actin, a rearward expansion of the actin network, and a reduction in retrograde flow speed. These results support the hypothesis that the principal role of cofilin in lamellipodia at steady state is to break down F-actin, control filament turnover, and regulate the rate of retrograde flow.

Growth cone travel in space and time: the cellular ensemble of cytoskeleton, adhesion, and membrane.

Growth cones, found at the tip of axonal projections, are the sensory and motile organelles of developing neurons that enable axon pathfinding and target recognition for precise wiring of the neural circuitry. To date, many families of conserved guidance molecules and their corresponding receptors have been identified that work in space and time to ensure billions of axons to reach their targets. Research in the past two decades has also gained significant insight into the ways in which growth cones translate extracellular signals into directional migration. This review aims to examine new progress toward understanding the cellular mechanisms underlying directional motility of the growth cone and to discuss questions that remain to be addressed. Specifically, we will focus on the cellular ensemble of cytoskeleton, adhesion, and membrane and examine how the intricate interplay between these processes orchestrates the directed movement of growth cones.

High-resolution quantification of focal adhesion spatiotemporal dynamics in living cells.

Focal adhesions (FAs) are macromolecular complexes that provide a linkage between the cell and its external environment. In a motile cell, focal adhesions change size and position to govern cell migration, through the dynamic processes of assembly and disassembly. To better understand the dynamic regulation of focal adhesions, we have developed an analysis system for the automated detection, tracking, and data extraction of these structures in living cells. This analysis system was used to quantify the dynamics of fluorescently tagged Paxillin and FAK in NIH 3T3 fibroblasts followed via Total Internal Reflection Fluorescence Microscopy (TIRF). High content time series included the size, shape, intensity, and position of every adhesion present in a living cell. These properties were followed over time, revealing adhesion lifetime and turnover rates, and segregation of properties into distinct zones. As a proof-of-concept, we show how a single point mutation in Paxillin at the Jun-kinase phosphorylation site Serine 178 changes FA size, distribution, and rate of assembly. This study provides a detailed, quantitative picture of FA spatiotemporal dynamics as well as a set of tools and methodologies for advancing our understanding of how focal adhesions are dynamically regulated in living cells. A full, open-source software implementation of this pipeline is provided at http://gomezlab.bme.unc.edu/tools.