Quantifying Protein Dynamics by Kymograph Analysis.

Time-lapse imaging of the subcellular localization and dynamic behavior of proteins is critical to understand their biological functions in cells. With the advent of various methodologies and computational tools, the precise tracking and quantification of protein spatiotemporal dynamics have become feasible. Kymograph analysis, in particular, has been extensively adopted for the quantitative assessment of proteins, vesicles, and organelle movements. However, conventional kymograph analysis, which is based on a single linear trajectory, may not comprehensively capture the complexity of proteins that alter their course during intracellular transport and activity. In this chapter, we introduced an advanced protocol for whole-cell kymograph analysis that allows for three-dimensional (3D) tracking of protein dynamics. This method was validated through the analysis of tip-focused endocytosis and exocytosis processes in growing tobacco pollen tubes by employing both the advanced whole-cell and classical kymograph methods. In addition, we enhanced this method by integrating pseudo-colored kymographs that enables the direct visualization of changes in protein fluorescence intensity with fluorescence recovery after photobleaching to advance our understanding of protein localization and dynamics. This comprehensive method offers a novel insight into the intricate dynamics of protein activity within the cellular context.

A Hands-on Guide to AmoePy - a Python-Based Software Package to Analyze Cell Migration Data.

Amoeboid cell motility is fundamental for a multitude of biological processes such as embryogenesis, immune responses, wound healing, and cancer metastasis. It is characterized by specific cell shape changes: the extension and retraction of membrane protrusions, known as pseudopodia. A common approach to investigate the mechanisms underlying this type of cell motility is to study phenotypic differences in the locomotion of mutant cell lines. To characterize such differences, methods are required to quantify the contour dynamics of migrating cells. AmoePy is a Python-based software package that provides tools for cell segmentation, contour detection as well as analyzing and simulating contour dynamics. First, a digital representation of the cell contour as a chain of nodes is extracted from each frame of a time-lapse microscopy recording of a moving cell. Then, the dynamics of these nodes-referred to as virtual markers-are tracked as the cell contour evolves over time. From these data, various quantities can be calculated that characterize the contour dynamics, such as the displacement of the virtual markers or the local stretching rate of the marker chain. Their dynamics is typically visualized in space-time plots, the so-called kymographs, where the temporal evolution is displayed for the different locations along the cell contour. Using AmoePy, you can straightforwardly create kymograph plots and videos from stacks of experimental bright-field or fluorescent images of motile cells. A hands-on guide on how to install and use AmoePy is provided in this chapter.

Measuring Retrograde Actin Flow in Neuronal Growth Cones.

Actin flow refers to the motion of the F-actin cytoskeleton and has been observed in many different cell types, especially in motile cells including neuronal growth cones. The direction of the actin flow is generally retrograde from the periphery toward the center of the cell. Actin flow can be harnessed for forward movement of the cell through substrate-cytoskeletal coupling; thus, a key function of actin flow is in cell locomotion. In this chapter, we illustrate three different methods of quantifying retrograde F-actin flow in growth cones derived from cultured Aplysia bag cell neurons. These methods include tracking the movement of surface marker beads as well as kymograph analysis of time-lapse sequences acquired by differential interference contrast (DIC) imaging or fluorescent speckle microscopy (FSM). Due to their large size, Aplysia neuronal growth cones are uniquely suited for these methods; however, they can also be applied to any other growth cones with clear F-actin-rich peripheral domains.

Vocal Vibration Opening Onset Position Abnormality in Patients With Adductor Spasmodic Dystonia by Using High-Speed Endoscopy.

OBJECTIVE: To analyze vocal fold vibration onset in patients with adductor laryngeal dystonia (ADLD) by analyzing vocal vibration opening onset position (VVOOP). STUDY DESIGN: Case-control study SETTING: A voice center. METHODS: Eleven patients with ADLD diagnosed in our voice center were enrolled in the ADLD group. Eleven healthy subjects matched by exact age and gender to the ADLD patients were selected as the control group. All subjects underwent laryngeal high-speed video endoscopy. VVOOP and its change were assessed by two otolaryngologists. The multiline video kymography was used to analyze the open quotient (OQ) and standard deviation of OQ. RESULTS: VVOOP had more than one position in 54.6% (6/11) of the patients with ADLD, which was higher than the control group (P < 0.05). VVOOP appeared in the front of the vocal fold in 54.6% (6/11) of patients with ADLD and in the back of the vocal fold in 81.8% (9/11) of patients with ADLD. VVOOP can be abnormal in 90.9% (10/11) of patients with ADLD, and the rate of VVOOP abnormality was higher than that of the control group (P < 0.05). Of 11, 6 (54.6%) patients with ADLD had a variable VVOOP; the variability rate of VVOOP was higher than that in the control group (P < 0.05). OQ and OQ standard deviation in the ADLD group were significantly greater than in the control group (P < 0.05). CONCLUSIONS: In patients with ADLD, vocal fold vibration was irregular, and VVOOP was abnormal and had a variable position and could reflect variability of the vocal vibration. LEVEL OF EVIDENCE: Level 4.

Quantifying Single and Dual Channel Live Imaging Data: Kymograph Analysis of Organelle Motility in Neurons.

Live imaging is commonly used to study dynamic processes in cells. Many labs carrying out live imaging in neurons use kymographs as a tool. Kymographs display time-dependent microscope data (time-lapsed images) in two-dimensional representations showing position vs. time. Extraction of quantitative data from kymographs, often done manually, is time-consuming and not standardized across labs. We describe here our recent methodology for quantitatively analyzing single color kymographs. We discuss the challenges and solutions of reliably extracting quantifiable data from single-channel kymographs. When acquiring in two fluorescent channels, the challenge becomes analyzing two objects that may co-traffic together. One must carefully examine the kymographs from both channels and decide which tracks are the same or try to identify the coincident tracks from an overlay of the two channels. This process is laborious and time consuming. The difficulty in finding an available tool for such analysis has led us to create a program to do so, called KymoMerge. KymoMerge semi-automates the process of identifying co-located tracks in multi-channel kymographs and produces a co-localized output kymograph that can be analyzed further. We describe our analysis, caveats, and challenges of two-color imaging using KymoMerge.

Assessment of Spindle Shape Control by Spindle Poleward Flux Measurements and FRAP Bulk Analysis.

In plants, the segregation of genetic material is achieved by an acentrosomal, mitotic spindle. This macromolecular machinery consists of different microtubule subpopulations and interacting proteins. The majority of what we know about the assembly and shape control of the mitotic spindle arose from vertebrate model systems. The dynamic properties of the individual tubulin polymers are crucial for the accurate assembly of the spindle array and are modulated by microtubule-associated motor and non-motor proteins. The mitotic spindle relies on a phenomenon called poleward microtubule flux that is critical to establish spindle shape, chromosome alignment, and segregation. This flux is under control of the non-motor microtubule-associated proteins and force-generating motors. Despite the large number of (plant-specific) kinesin motor proteins expressed during mitosis, their mitotic roles remain largely elusive. Moreover, reports on mitotic spindle formation and shape control in higher plants are scarce. In this chapter, an overview of the basic principles and methods concerning live imaging of prometa- and metaphase spindles and the analysis of spindle microtubule flux using fluorescence recovery after photobleaching is provided.

Employing Live-Cell Imaging to Study Motor-Mediated Transport.

Microtubule-based transport is a highly regulated process, requiring kinesin and/or dynein motors, a multitude of motor-associated regulatory proteins including activating adaptors and scaffolding proteins, and microtubule tracks that also provide regulatory cues. While in vitro studies are invaluable, fully replicating the physiological conditions under which motility occurs in cells is not yet possible. Here, we describe two methods that can be employed to study motor-based transport and motor regulation in a cellular context. Live-cell imaging of organelle transport in neurons leverages the uniform polarity of microtubules in axons to better understand the factors regulating microtubule-based motility. Peroxisome recruitment assays allow users to examine the net effect of motors and motor-regulatory proteins on organelle distribution. Together, these methods open the door to motility experiments that more fully interrogate the complex cellular environment.

The conformations and basal conformational dynamics of translocation factor SecDF vary with translocon SecYEG interaction.

The general secretory, or Sec, system is a primary protein export pathway from the cytosol of Escherichia coli and all eubacteria. Integral membrane protein complex SecDF is a translocation factor that enhances polypeptide secretion, which is driven by the Sec translocase, consisting of translocon SecYEG and ATPase SecA. SecDF is thought to utilize a proton gradient to effectively pull precursor proteins from the cytoplasm into the periplasm. Working models have been developed to describe the structure and function of SecDF, but important mechanistic questions remain unanswered. Atomic force microscopy (AFM) is a powerful technique for studying the dynamics of single-molecule systems including membrane proteins in near-native conditions. The sharp tip of the AFM provides direct access to membrane-external protein conformations. Here, we acquired AFM images and kymographs (∼100 ms resolution) to visualize SecDF protrusions in near-native supported lipid bilayers and compared the experimental data to simulated AFM images based on static structures. When studied in isolation, SecDF exhibited a stable and compact conformation close to the lipid bilayer surface, indicative of a resting state. Interestingly, upon SecYEG introduction, we observed changes in both SecDF conformation and conformational dynamics. The population of periplasmic protrusions corresponding to an intermediate form of SecDF, which is thought to be active in precursor protein handling, increased more than ninefold. In conjunction, our dynamics measurements revealed an enhancement in the transition rate between distinct SecDF conformations when the translocon was present. Together, this work provides a novel vista of basal-level SecDF conformational dynamics in near-native conditions.

Live Imaging and Quantitative Analysis of Organelle Transport in Sensory Neurons of Aplysia Californica.

Axonal transport moves proteins, RNAs, and organelles between the soma and synapses to support synaptic function and activity-dependent changes in synaptic strength. This transport is impaired in several neurodegenerative disorders such as Alzheimer's disease. Thus, it is critical to understand the regulation and underlying mechanisms of the transport process. Aplysia californica provides a powerful experimental system for studying the interplay between synaptic activity and transport because its defined synaptic circuits can be built in-vitro. Advantages include precise pre- and postsynaptic manipulation, and high-resolution imaging of axonal transport. Here, we describe methodologies for the quantitative analysis of axonal transport in Aplysia sensory neurons.

Automated capillary flow segmentation and mapping for nailfold video capillaroscopy.

OBJECTIVE: This study aimed to develop an automated image analysis method for segmentation and mapping of capillary flow dynamics captured using nailfold video capillaroscopy (NVC). Methods were applied to compare capillary flow structures and dynamics between young and middle-aged healthy controls. METHODS: NVC images were obtained in a resting state, and a region of the vessel in the image was extracted using a conventional U-Net neural network. The approximate length, diameter, and radius of the curvature were calculated automatically. Flow speed and its fluctuation over time were mapped using the Radon transform and frequency spectrum analysis from the kymograph image created along the vessel's centerline. RESULTS: The diameter of the curve segment (14.4 µm and 13.0 µm) and the interval of two straight segments (13.7 µm and 32.1 µm) of young and middle-aged subjects, respectively, were significantly different. Faster flow was observed in older subjects (0.48 mm/s) than in younger subjects (0.26 mm/s). The power spectral analysis revealed a significant correlation between the high-frequency power spectrum and the flow speed. CONCLUSIONS: The present method allows a spatiotemporal characterization of capillary morphology and flow dynamics with NVC, allowing a wide application such as large-scale health assessment.

The dendritic ERGIC: Microtubule and actin cytoskeletons participate in stop-and-go movement of mobile carriers between stable structures.

The endoplasmic reticulum (ER)-to-Golgi intermediate compartment (ERGIC) is a membranous organelle that mediates protein transport between the ER and the Golgi apparatus. In neurons, clusters of these vesiculotubular structures are situated throughout the cell in proximity to the ER, passing cargo to the cis-Golgi cisternae, located mainly in the perinuclear region. Although ERGIC markers have been identified in neurons, the distribution and dynamics of neuronal ERGIC structures have not been characterized yet. Here, we show that long-distance ERGIC transport occurs via an intermittent mechanism in dendrites, with mobile elements moving between stationary structures. Slow and fast live-cell imaging have captured stable ERGIC structures remaining in place over long periods of time, as well as mobile ERGIC structures advancing very short distances along dendrites. These short distances have been consistent with the lengths between the stationary ERGIC structures. Kymography revealed ERGIC elements that moved intermittently, emerging from and fusing with stationary ERGIC structures. Interestingly, this movement apparently depends not only on the integrity of the microtubule cytoskeleton, as previously reported, but on the actin cytoskeleton as well. Our results indicate that the dendritic ERGIC has a dual nature, with both stationary and mobile structures. The neural ERGIC network transports proteins via a stop-and-go movement in which both the microtubule and the actin cytoskeletons participate.

Deciphering the dynamics of lamellipodium in a fish keratocytes model.

Motile cells depend on an intricate network of feedback loops that are essential in driving cell movement. Integrin-based focal adhesions (FAs) along with actin are the two key factors that mediate such motile behaviour. Together, they generate excitable dynamics that are essential for forming protrusions at the leading edge of the cell and, in certain cases, traveling waves along the membrane. A partial differential equation (PDE) model of a self-organizing lamellipodium in crawling keratocytes has been previously developed to understand how the three spatiotemporal patterns of activity observed in such cells, namely, stalling, waving and smooth motility, are produced. The model consisted of three key variables: the density of barbed actin filaments, newly formed FAs called nascent adhesions (NAs) and VASP, an anti-capping protein that gets sequestered by NAs during maturation. Using parameter sweeping techniques, the distinct regimes of behaviour associated with the three activity patterns were identified. In this study, we convert the PDE model into an ordinary differential equation (ODE) model to examine its excitability properties and determine all the patterns of activity exhibited by this system. Our results reveal that there are two additional regimes not previously identified, including bistability and oscillatory-like type IV excitability (generated by three steady states and their manifolds, rather than limit cycles). These regimes are also present in the PDE model. Applying slow-fast analysis on the ODE model shows that it exhibits a canard explosion through a folded-saddle and that rough motility seen in keratocytes is likely due to noise-dependent motility governed by dynamics near the interface of bistability and type IV excitability. The two parameter bifurcation suggests that the increase in the proportion of rough motion is due to a shift in activity towards the bistable and type IV excitable regimes induced by a decrease in NA maturation rate. Our results thus provide important insight into how microscopic mechanical effects are integrated to produce the observed modes of motility.

Spatiotemporal Analysis of Caveolae Dynamics Using Total Internal Reflection Fluorescence Microscopy.

Total internal reflection fluorescence microscopy enables to analyze the localizations and dynamics of cellular events that occur at or near the plasma membrane. Total internal reflection fluorescence microscopy exclusively illuminates molecules in the close vicinity of the glass surface, thereby reducing background fluorescence and enabling observation of the plasma membrane in the glass-attached cells with a high signal-to-noise ratio. Here, we describe the application of total internal reflection fluorescence microscopy to analyze the dynamics of caveolae, which play essential physiological functions, including membrane tension buffering, endocytosis, and signaling at the plasma membrane.

Processing TIRF Microscopy Images to Characterize the Dynamics and Morphology of Bacterial Actin-Like Assemblies.

Total internal reflection fluorescence (TIRF) microscopy allows the visualization of the dynamic membrane-associated actin-like MreB filaments in live bacterial cells with high temporal resolution. This chapter describes computerized analysis methods to quantitatively characterize the dynamics and morphological properties of MreB assemblies. These include how to (1) segment bacterial cells, (2) perform single-particle tracking (SPT) of MreB filamentous structures, (3) classify their dynamic modes using mean squared displacement (MSD) analysis, and (4) measure their dimensions and orientation.

Measuring microtubule dynamics.

Microtubules are key players in cellular self-organization, acting as structural scaffolds, cellular highways, force generators and signalling platforms. Microtubules are polar filaments that undergo dynamic instability, i.e. transition between phases of growth and shrinkage. This allows microtubules to explore the inner space of the cell, generate pushing and pulling forces and remodel themselves into arrays with different geometry and function such as the mitotic spindle. To do this, eukaryotic cells employ an arsenal of regulatory proteins to control microtubule dynamics spatially and temporally. Plants and microorganisms have developed secondary metabolites that perturb microtubule dynamics, many of which are in active use as cancer chemotherapeutics and anti-inflammatory drugs. Here, we summarize the methods used to visualize microtubules and to measure the parameters of dynamic instability to study both microtubule regulatory proteins and the action of small molecules interfering with microtubule assembly and/or disassembly.

A simple and efficient toolset for analysing mitochondrial trafficking in neuronal cells.

Mitochondria are crucial for cells, supplying up to 90% of the energy requirements for neurons. Their correct localisation is crucial and ensured by a transport system. Mitochondrial trafficking in neurons is particularly critical, because mitochondria must leave the soma and travel along the axon and dendritic network to facilitate neuronal function. Abnormal mitochondrial trafficking has been reported in several neurological disorders, therefore the ability to quantify and analyse mitochondrial trafficking is vital to improving our understanding of their pathogenesis. Commercial software currently lacks an automated approach for performing such quantitation. Here we demonstrate the development of the Mitochondrial Trafficking and Distribution (MiTrakD) analysis toolset, which consists of simple and free-to-use instructions for mitochondrial trafficking analysis using time-lapse microscopy. MiTrakD utilises existing Fiji (ImageJ) tools for semi-automated, fast and efficient analysis of mitochondrial trafficking and distribution, including velocity, abundance, localisation and distance travelled in neurons. We document MiTrakD's efficiency and accuracy by analysing mitochondrial trafficking using two-dimensional fluorescence images of cortical neurons of wild type mice after 6 days (DIV6), 10 days (DIV10) and 14 days (DIV14) of in vitro incubation. Using MiTrakD we have demonstrated that neurons at all developmental stages exhibited the same percentage of mobile mitochondria, all of which travel in equidistance. Interestingly, the mitochondria in neurons at DIV10 were in greater abundance and were faster than those at DIV6 and DIV14. We can also conclude that MiTrakD is more efficient than manual analysis and is an accurate and reliable tool for performing mitochondrial trafficking analysis in neuronal cells.

Imaging of Endothelial Cell Dynamic Behavior in Zebrafish.

In recent years, use of the zebrafish embryo as a model organism to study vascular development in vivo has provided valuable insights into the genetic and cellular events shaping the embryonic vasculature. In this chapter, we aim to present the methods for the measurement of some of the most commonly investigated dynamic parameters in endothelial cells during developmental angiogenesis, namely, migration speed and acceleration, filopodia extension, front-rear polarity, cell cycle progression, membrane deformations, and junctional rearrangements. We also offer suggestions on how to deal with the most common imaging and quantifications challenges faced when acquiring and quantifying endothelial cell dynamic behavior in vivo.We intend this section to serve as an experience-based imaging primer for scientists interested in endothelial cell imaging in the zebrafish embryo.

Axonal neurofilaments exhibit frequent and complex folding behaviors.

Neurofilaments are flexible cytoskeletal polymers that are capable of folding and unfolding between their bouts of bidirectional movement along axons. Here we present a detailed characterization of this behavior in cultured neurons using kymograph analysis with approximately 30 ms temporal resolution. We analyzed 781 filaments ranging from 0.6-42 µm in length. We observed complex behaviors including pinch folds, hairpin folds, orientation changes (flips), and occasional severing and annealing events. On average, the filaments spent approximately 40% of their time in some sort of folded configuration. A small proportion of filaments (4%) moved while folded, but most (96%) moved in an outstretched configuration. Collectively, our observations suggest that motors may interact with neurofilaments at multiple points along their length, but preferentially at their ends. In addition, the prevalence of neurofilament folding and the tendency of neurofilaments to straighten out when they move, suggest that an important function of the movement of these polymers in axons may be to maintain them in an outstretched and longitudinally co-aligned configuration. Thus, neurofilament movement may function as much to organize these polymers as to move them, and this could explain why they spend so much time engaged in apparently unproductive bidirectional movement.

Kymograph analysis with high temporal resolution reveals new features of neurofilament transport kinetics.

We have used kymograph analysis combined with edge detection and an automated computational algorithm to analyze the axonal transport kinetics of neurofilament polymers in cultured neurons at 30 ms temporal resolution. We generated 301 kymographs from 136 movies and analyzed 726 filaments ranging from 0.6 to 42 µm in length, representing ∼37,000 distinct moving and pausing events. We found that the movement is even more intermittent than previously reported and that the filaments undergo frequent, often transient, reversals which suggest that they can engage simultaneously with both anterograde and retrograde motors. Average anterograde and retrograde bout velocities (0.9 and 1.2 µm s-1 , respectively) were faster than previously reported, with maximum sustained bout velocities of up to 6.6 and 7.8 µm s-1 , respectively. Average run lengths (∼1.1 µm) and run times (∼1.4 s) were in the range reported for molecular motor processivity in vitro, suggesting that the runs could represent the individual processive bouts of the neurofilament motors. Notably, we found no decrease in run velocity, run length or run time with increasing filament length, which suggests that either the drag on the moving filaments is negligible or that longer filaments recruit more motors.