Direct observation of aggregate-triggered selective autophagy in human cells.

Degradation of aggregates by selective autophagy is important as damaged proteins may impose a threat to cellular homeostasis. Although the core components of the autophagy machinery are well characterized, the spatiotemporal regulation of many selective autophagy processes, including aggrephagy, remains largely unexplored. Furthermore, because most live-cell imaging studies have so far focused on starvation-induced autophagy, little is known about the dynamics of aggrephagy. Here, we describe the development and application of the mKeima-PIM assay, which enables live-cell observation of autophagic turnover and degradation of inducible protein aggregates in conjunction with key autophagy players. This allowed us to quantify the relative timing and duration of different steps of aggrephagy in human cells and revealed the short-lived nature of the autophagosome. The assay furthermore showed the spatial distribution of omegasome formation, highlighting that autophagy initiation is directly instructed by the cargo. Moreover, we found that nascent autophagosomes mostly remain immobile until acidification occurs. Thus, our assay provides new insights into the spatiotemporal regulation and dynamics of aggrephagy. This article has an associated First Person interview with the first author of the paper.

HSV-1 Glycoproteins Are Delivered to Virus Assembly Sites Through Dynamin-Dependent Endocytosis.

Herpes simplex virus-1 (HSV-1) is a large enveloped DNA virus that belongs to the family of Herpesviridae. It has been recently shown that the cytoplasmic membranes that wrap the newly assembled capsids are endocytic compartments derived from the plasma membrane. Here, we show that dynamin-dependent endocytosis plays a major role in this process. Dominant-negative dynamin and clathrin adaptor AP180 significantly decrease virus production. Moreover, inhibitors targeting dynamin and clathrin lead to a decreased transport of glycoproteins to cytoplasmic capsids, confirming that glycoproteins are delivered to assembly sites via endocytosis. We also show that certain combinations of glycoproteins colocalize with each other and with the components of clathrin-dependent and -independent endocytosis pathways. Importantly, we demonstrate that the uptake of neutralizing antibodies that bind to glycoproteins when they become exposed on the cell surface during virus particle assembly leads to the production of non-infectious HSV-1. Our results demonstrate that transport of viral glycoproteins to the plasma membrane prior to endocytosis is the major route by which these proteins are localized to the cytoplasmic virus assembly compartments. This highlights the importance of endocytosis as a major protein-sorting event during HSV-1 envelopment.

Current Methods and Pipelines for Image-Based Quantitation of Nuclear Shape and Nuclear Envelope Abnormalities.

Any given cell type has an associated "normal" nuclear morphology, which is important to maintain proper cellular functioning and safeguard genomic integrity. Deviations from this can be indicative of diseases such as cancer or premature aging syndrome. To accurately assess nuclear abnormalities, it is important to use quantitative measures of nuclear morphology. Here, we give an overview of several nuclear abnormalities, including micronuclei, nuclear envelope invaginations, blebs and ruptures, and review the current methods used for image-based quantification of these abnormalities. We discuss several parameters that can be used to quantify nuclear shape and compare their outputs using example images. In addition, we present new pipelines for quantitative analysis of nuclear blebs and invaginations. Quantitative analyses of nuclear aberrations and shape will be important in a wide range of applications, from assessments of cancer cell anomalies to studies of nucleus deformability under mechanical or other types of stress.

Probing aggrephagy using chemically-induced protein aggregates.

Selective types of autophagy mediate the clearance of specific cellular components and are essential to maintain cellular homeostasis. However, tools to directly induce and monitor such pathways are limited. Here we introduce the PIM (particles induced by multimerization) assay as a tool for the study of aggrephagy, the autophagic clearance of aggregates. The assay uses an inducible multimerization module to assemble protein clusters, which upon induction recruit ubiquitin, p62, and LC3 before being delivered to lysosomes. Moreover, use of a dual fluorescent tag allows for the direct observation of cluster delivery to the lysosome. Using flow cytometry and fluorescence microscopy, we show that delivery to the lysosome is partially dependent on p62 and ATG7. This assay will help in elucidating the spatiotemporal dynamics and control mechanisms underlying aggregate clearance by the autophagy-lysosomal system.

Myosin-V Induces Cargo Immobilization and Clustering at the Axon Initial Segment.

The selective transport of different cargoes into axons and dendrites underlies the polarized organization of the neuron. Although it has become clear that the combined activity of different motors determines the destination and selectivity of transport, little is known about the mechanistic details of motor cooperation. For example, the exact role of myosin-V in opposing microtubule-based axon entries has remained unclear. Here we use two orthogonal chemically-induced heterodimerization systems to independently recruit different motors to cargoes. We find that recruiting myosin-V to kinesin-propelled cargoes at approximately equal numbers is sufficient to stall motility. Kinesin-driven cargoes entering the axon were arrested in the axon initial segment (AIS) upon myosin-V recruitment and accumulated in distinct actin-rich hotspots. Importantly, unlike proposed previously, myosin-V did not return these cargoes to the cell body, suggesting that additional mechanism are required to establish cargo retrieval from the AIS.

Three-Step Model for Polarized Sorting of KIF17 into Dendrites.

Kinesin and dynein motors drive bidirectional cargo transport along microtubules and have a critical role in polarized cargo trafficking in neurons [1, 2]. The kinesin-2 family protein KIF17 is a dendrite-specific motor protein and has been shown to interact with several dendritic cargoes [3-7]. However, the mechanism underlying the dendritic targeting of KIF17 remains poorly understood [8-11]. Using live-cell imaging combined with inducible trafficking assays to directly probe KIF17 motor activity in living neurons, we found that the polarized sorting of KIF17 to dendrites is regulated in multiple steps. First, cargo binding of KIF17 relieves autoinhibition and initiates microtubule-based cargo transport. Second, KIF17 does not autonomously target dendrites, but enters the axon where the actin cytoskeleton at the axon initial segment (AIS) prevents KIF17 vesicles from moving further into the axon. Third, dynein-based motor activity is able to redirect KIF17-coupled cargoes into dendrites. We propose a three-step model for polarized targeting of KIF17, in which the collective function of multiple motor teams is required for proper dendritic sorting.

Design and self-assembly of simple coat proteins for artificial viruses.

Viruses are among the simplest biological systems and are highly effective vehicles for the delivery of genetic material into susceptible host cells. Artificial viruses can be used as model systems for providing insights into natural viruses and can be considered a testing ground for developing artificial life. Moreover, they are used in biomedical and biotechnological applications, such as targeted delivery of nucleic acids for gene therapy and as scaffolds in material science. In a natural setting, survival of viruses requires that a significant fraction of the replicated genomes be completely protected by coat proteins. Complete protection of the genome is ensured by a highly cooperative supramolecular process between the coat proteins and the nucleic acids, which is based on reversible, weak and allosteric interactions only. However, incorporating this type of supramolecular cooperativity into artificial viruses remains challenging. Here, we report a rational design for a self-assembling minimal viral coat protein based on simple polypeptide domains. Our coat protein features precise control over the cooperativity of its self-assembly with single DNA molecules to finally form rod-shaped virus-like particles. We confirm the validity of our design principles by showing that the kinetics of self-assembly of our virus-like particles follows a previous model developed for tobacco mosaic virus. We show that our virus-like particles protect DNA against enzymatic degradation and transfect cells with considerable efficiency, making them promising delivery vehicles.