Free diffusion of PI(4,5)P2 in the plasma membrane in the presence of high density effector protein complexes

The lipid phosphatidyl-D-myo-inositol-4,5-bisphosphate [PI(4,5)P2] is a master regulator of plasma membrane (PM) function. It engages effector proteins that regulate diverse traffic, transport, signaling and cytoskeletal processes that define PM structure and function. How a single class of lipid molecules independently regulate so many parallel processes remains an open question. We tested the hypothesis that spatially segregated pools of PI(4,5)P2 are associated with, and thus reserved for regulation of, different functional complexes in the PM. The mobility of PI(4,5)P2 in the membrane was measured using lipid biosensors by single particle tracking photoactivation localization microscopy (sptPALM). We found that PI(4,5)P2, and several other classes of inner PM lipids, diffuse rapidly at approximately 0.3 microns squared per second with largely Brownian motion, although they spend approximately a third of their time diffusing much more slowly. Surprisingly, areas of the PM occupied by PI(4,5)P2-dependent complexes, such endoplasmic-reticulum:PM contact sites, clathrin-coated structures, and several actin cytoskeletal elements including focal adhesions, did not cause a change in PI(4,5)P2 lateral mobility. Only the spectrin and septin cytoskeletons were observed to produce a slowing of PI(4,5)P2 diffusion. We conclude that even structures with high densities of PI(4,5)P2-engaging effector proteins, such as clathrin coated pits and focal adhesions, do not corral free PI(4,5)P2, questioning a role for spatially segregated PI(4,5)P2 pools in organizing and regulating parallel PM functions.

Nanometer-Resolution Long-term Tracking of Single Cargos Reveals Dynein Motor Mechanisms

Cytoplasmic dynein is essential for intracellular transport, but because of its complexity, we still do not fully understand how this 1.5 megadalton protein works. Here, we used novel optical probes that enable single-particle tracking (SPT) of individual cargos transported by dynein motors in live neurons over 900 μm. Analyses using the Fluctuation Theorem (FT) showed that the number of dynein molecules switches between 1-5 motors during the transport. Clearly resolved single-molecular steps revealed that the dwell times between individual steps were accurately described by an enzymatic cycle dominated by two equal and thermally-activated rate constants. Based on these data, we propose a new molecular model whereby each step requires the hydrolysis of 2 ATPs. The model is consistent with extensive structural, single-molecule and biochemical measurements.

Revising the Role of Cortical Cytoskeleton during Secretion: Actin and Myosin XI Function in Vesicle Tethering

In plants, secretion of cell wall components and membrane proteins plays a fundamental role in growth and development as well as survival in diverse environments. Exocytosis, as the last step of the secretory trafficking pathway, is a highly ordered and precisely controlled process involving tethering, docking, and fusion of vesicles at the plasma membrane (PM) for cargo delivery. Although the exocytic process and machinery are well characterized in yeast and animal models, the molecular players and specific molecular events that underpin late stages of exocytosis in plant cells remain largely unknown. Here, by using the delivery of functional, fluorescent-tagged cellulose synthase (CESA) complexes (CSCs) to the PM as a model system for secretion, as well as single-particle tracking in living cells, we describe a quantitative approach for measuring the frequency of vesicle tethering events. Genetic and pharmacological inhibition of cytoskeletal function, reveal that the initial vesicle tethering step of exocytosis is dependent on actin and myosin XI. In contrast, treatments with the microtubule inhibitor, oryzalin, did not significantly affect vesicle tethering or fusion during CSC exocytosis but caused a minor increase in transient or aborted tethering events. With data from this new quantitative approach and improved spatiotemporal resolution of single particle events during secretion, we generate a revised model for the role of the cortical cytoskeleton in CSC trafficking.

SHOT-R: A next generation algorithm for particle kinematics analysis

Analysis of live-imaging experiments is crucial to decipher a plethora of cellular mechanisms within physiological and pathological contexts. Kymograph, i.e. graphical representations of particle spatial position over time, and single particle tracking (SPT) are the currently available tools to extract information on particle transport and velocity. However, the spatiotemporal approximation applied in particle trajectory reconstruction with those methods intrinsically prevents an accurate analysis of particle kinematics and of instantaneous behaviours. Here, we present SHOT-R, a novel numerical method based on polynomial reconstruction of 4D (3D+time) particle trajectories. SHOT-R, contrary to other tools, computes bona fide instantaneous and directional velocity, and acceleration. Thanks to its high order continuous reconstruction it allows, for the first time, kinematics analysis of co-trafficked particles. Overall, SHOT-R is a novel, versatile, and physically reliable numerical method that achieves all-encompassing particle kinematics studies at unprecedented accuracy on any live-imaging experiment where the spatiotemporal coordinates can be retrieved.

What is motion? Recent advances in the study of molecular movement patterns of the peptidoglycan synthesis machines

How proteins move through space and time is a fundamental question in biology. While great strides have been made towards a mechanistic understanding of protein movement, many questions remain. We discuss the biological implications of motion in the context of the peptidoglycan (PG) synthesis machines. We review systems in several bacteria, including Escherichia coli , Bacillus subtilis , and Streptococcus pneumoniae , and present a comprehensive view of our current knowledge regarding movement dynamics. Discrepancies are also addressed since “one size does not fit all”. For bacteria to divide, new PG is synthesized and incorporated into the growing cell wall by complex multi-protein nanomachines consisting of PG synthases (transglycosylases [TG] and/or transpeptidases [TP]) as well as a variety of regulators and cytoskeletal factors. Advances in imaging capabilities and labeling methods have revealed that these machines are not static but rather circumferentially transit the cell via directed motion perpendicular to the long axis of model rod-shaped bacteria such as E. coli and B. subtilis . The enzymatic activity of the TG:TPs drives motion in some species, while motion is mediated by FtsZ treadmilling in others. In addition, both directed and diffusive motion of the PG synthases has been observed using single particle tracking technology. Here, we examine the biological role of diffusion regarding transit. Lastly, findings regarding the monofunctional transglycosylases (RodA and FtsW) as well as the Class A PG synthases are discussed. This minireview serves to showcase recent advances, broach mechanistic unknowns, and stimulate future areas of study.

Infection-induced chromatin modifications facilitate translocation of herpes simplex virus capsids to the inner nuclear membrane

Herpes simplex virus capsids are assembled and packaged in the nucleus and move by diffusion through the nucleoplasm to the nuclear envelope for egress. Analyzing their motion provides conclusions not only on capsid transport but also on the properties of the nuclear environment during infection. We utilized live-cell imaging and single-particle tracking to characterize capsid motion relative to the host chromatin. The data indicate that as the chromatin was marginalized toward the nuclear envelope it presented a restrictive barrier to the capsids. However, later in infection this barrier became more permissive and the probability of capsids to enter the chromatin increased. Thus, although chromatin marginalization initially restricted capsid transport to the nuclear envelope, a structural reorganization of the chromatin counteracted that to promote capsid transport later. Analyses of capsid motion revealed that it was subdiffusive, and that the diffusion coefficients were lower in the chromatin than in regions lacking chromatin. In addition, the diffusion coefficient in both regions increased during infection. Throughout the infection, the capsids were never enriched at the nuclear envelope, which suggests that instead of nuclear export the transport through the chromatin is the rate-limiting step for the nuclear egress of capsids. This provides motivation for further studies by validating the importance of intranuclear transport to the life cycle of HSV-1.

CRISPR Cas13-based tools to track and manipulate endogenous telomeric repeat-containing RNAs in living cells

TERRA, TElomeric Repeat-containing RNA, is a long non-coding RNA transcribed from telomeres. Emerging evidence indicates that TERRA regulates telomere maintenance and chromosome end protection in normal and cancerous cells. However, the mechanism of how TERRA contributes to telomere functions is still unclear, partially owing to the shortage of approaches to track and manipulate endogenous TERRA molecules in live cells. Here, we developed a method to visualize TERRA in live cells via a combination of CRISPR Cas13 RNA labeling and Suntag technology. Single-particle tracking reveals that TERRA foci undergo anomalous diffusion in a manner that depends on the timescale and telomeric localization. Furthermore, we used a chemically-induced protein dimerization system to manipulate TERRA subcellular localization in live cells. Overall, our approaches to monitor and control TERRA locations in live cells provide powerful tools to better understand its roles in telomere maintenance and genomic integrity.

Revising the Role of Cortical Cytoskeleton During Secretion: Actin and Myosin XI Function in Vesicle Tethering

In plants, secretion of cell wall components and membrane proteins plays a fundamental role in growth and development as well as survival in diverse environments. Exocytosis, as the last step of the secretory trafficking pathway, is a highly ordered and precisely controlled process involving tethering, docking, and fusion of vesicles at the plasma membrane (PM) for cargo delivery. Although the exocytic process and machinery are well characterized in yeast and animal models, the molecular players and specific molecular events that underpin late stages of exocytosis in plant cells remain largely unknown. Here, by using the delivery of functional, fluorescent-tagged cellulose synthase (CESA) complexes (CSCs) to the PM as a model system for secretion, as well as single-particle tracking in living cells, we describe a quantitative approach for measuring the frequency of vesicle tethering events. Genetic and pharmacological inhibition of cytoskeletal function, reveal that the initial vesicle tethering step of exocytosis is dependent on actin and myosin XI. In contrast, treatments with the microtubule inhibitor, oryzalin, did not significantly affect vesicle tethering or fusion during CSC exocytosis but caused a minor increase of transient or aborted tethering events. With data from this new quantitative approach and improved spatiotemporal resolution of single particle events during secretion, we generate a revised model for the role of the cortical cytoskeleton in CSC trafficking.

EGFR transactivates RON to drive oncogenic crosstalk

Crosstalk between different receptor tyrosine kinases (RTKs) is thought to drive oncogenic signaling and allow therapeutic escape. EGFR and RON are two such RTKs from different subfamilies, which engage in crosstalk through unknown mechanisms. We combined high-resolution imaging with biochemical and mutational studies to ask how EGFR and RON communicate. EGF stimulation promotes EGFR-dependent phosphorylation of RON, but ligand stimulation of RON does not trigger EGFR phosphorylation – arguing that crosstalk is unidirectional. Nanoscale imaging reveals association of EGFR and RON in common plasma membrane microdomains. Two-color single particle tracking captured formation of complexes between RON and EGF-bound EGFR. Our results further show that RON is a substrate for EGFR kinase, and that transactivation of RON requires formation of a signaling competent EGFR dimer. These results support a role for direct EGFR/RON interactions in propagating crosstalk, such that EGF-stimulated EGFR phosphorylates RON to activate RON-directed signaling.