Clathrin-mediated endocytosis is independent of actin dynamics in many circumstances but requires actin polymerization in others. We show that membrane tension determines the actin dependence of ...clathrin-coat assembly. As found previously, clathrin assembly supports formation of mature coated pits in the absence of actin polymerization on both dorsal and ventral surfaces of non-polarized mammalian cells, and also on basolateral surfaces of polarized cells. Actin engagement is necessary, however, to complete membrane deformation into a coated pit on apical surfaces of polarized cells and, more generally, on the surface of any cell in which the plasma membrane is under tension from osmotic swelling or mechanical stretching. We use these observations to alter actin dependence experimentally and show that resistance of the membrane to propagation of the clathrin lattice determines the distinction between 'actin dependent and 'actin independent'. We also find that light-chain-bound Hip1R mediates actin engagement. These data thus provide a unifying explanation for the role of actin dynamics in coated-pit budding.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
A central hurdle in developing small interfering RNAs (siRNAs) as therapeutics is the inefficiency of their delivery across the plasma and endosomal membranes to the cytosol, where they interact with ...the RNA interference machinery. With the aim of improving endosomal release, a poorly understood and inefficient process, we studied the uptake and cytosolic release of siRNAs, formulated in lipoplexes or lipid nanoparticles, by live-cell imaging and correlated it with knockdown of a target GFP reporter. siRNA release occurred invariably from maturing endosomes within ~5-15 min of endocytosis. Cytosolic galectins immediately recognized the damaged endosome and targeted it for autophagy. However, inhibiting autophagy did not enhance cytosolic siRNA release. Gene knockdown occurred within a few hours of release and required <2,000 copies of cytosolic siRNAs. The ability to detect cytosolic release of siRNAs and understand how it is regulated will facilitate the development of rational strategies for improving the cytosolic delivery of candidate drugs.
The shape and total surface of a cell and its daughters change during mitosis. Many cells round up during prophase and metaphase and reacquire their extended and flattened shape during cytokinesis. ...How does the total area of plasma membrane change to accommodate these morphological changes and by what mechanism is control of total membrane area achieved? Using single-cell imaging methods, we have found that the amount of plasma membrane in attached cells in culture decreases at the beginning of mitosis and recovers rapidly by the end. Clathrin-based endocytosis is normal throughout all phases of cell division, whereas recycling of internalized membranes back to the cell surface slows considerably during the rounding up period and resumes at the time at which recovery of cell membrane begins. Interference with either one of these processes by genetic or chemical means impairs cell division. The total cell-membrane area recovers even in the absence of a functional Golgi apparatus, which would be needed for export of newly synthesized membrane lipids and proteins. We propose a mechanism by which modulation of endosomal recycling controls cell area and surface expression of membrane-bound proteins during cell division.
Many viruses that enter cells by clathrin-dependent endocytosis are significantly larger than the dimensions of a typical clathrin-coated vesicle. The mechanisms by which viruses co-opt the clathrin ...machinery for efficient internalization remain uncertain. Here we examined how clathrin-coated vesicles accommodate vesicular stomatitis virus (VSV) during its entry into cells. Using high-resolution imaging of the internalization of single viral particles into cells expressing fluorescent clathrin and adaptor molecules, we show that VSV enters cells through partially clathrin-coated vesicles. We found that on average, virus-containing vesicles contain more clathrin and clathrin adaptor molecules than conventional vesicles, but this increase is insufficient to permit full coating of the vesicle. We further show that virus-containing vesicles depend upon the actin machinery for their internalization. Specifically, we found that components of the actin machinery are recruited to virus-containing vesicles, and chemical inhibition of actin polymerization trapped viral particles in vesicles at the plasma membrane. By analysis of multiple independent virus internalization events, we show that VSV induces the nucleation of clathrin for its uptake, rather than depending upon random capture by formation of a clathrin-coated pit. This work provides new mechanistic insights into the process of virus internalization as well as uptake of unconventional cargo by the clathrin-dependent endocytic machinery.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Clathrin is the scaffold of a conserved molecular machinery that has evolved to capture membrane patches, which then pinch off to become traffic carriers. These carriers are the principal vehicles of ...receptor-mediated endocytosis and are the major route of traffic from plasma membrane to endosomes. We report here the use of in vivo imaging data, obtained from spinning disk confocal and total internal reflection fluorescence microscopy, to distinguish between two modes of endocytic clathrin coat formation, which we designate as "coated pits" and "coated plaques." Coated pits are small, rapidly forming structures that deform the underlying membrane by progressive recruitment of clathrin, adaptors, and other regulatory proteins. They ultimately close off and bud inward to form coated vesicles. Coated plaques are longer-lived structures with larger and less sharply curved coats; their clathrin lattices do not close off, but instead move inward from the cell surface shortly before membrane fission. Local remodeling of actin filaments is essential for the formation, inward movement, and dissolution of plaques, but it is not required for normal formation and budding of coated pits in the cells we have studied. We conclude that there are at least two distinct modes of clathrin coat formation at the plasma membrane--classical coated pits and coated plaques--and that these two assemblies interact quite differently with other intracellular structures.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
How proteins control the biogenesis of cellular lipid droplets (LDs) is poorly understood. Using
and human cells, we show here that seipin, an ER protein implicated in LD biology, mediates a discrete ...step in LD formation-the conversion of small, nascent LDs to larger, mature LDs. Seipin forms discrete and dynamic foci in the ER that interact with nascent LDs to enable their growth. In the absence of seipin, numerous small, nascent LDs accumulate near the ER and most often fail to grow. Those that do grow prematurely acquire lipid synthesis enzymes and undergo expansion, eventually leading to the giant LDs characteristic of seipin deficiency. Our studies identify a discrete step of LD formation, namely the conversion of nascent LDs to mature LDs, and define a molecular role for seipin in this process, most likely by acting at ER-LD contact sites to enable lipid transfer to nascent LDs.
Adding to the super-resolution arsenal
Structured illumination microscopy (SIM) uses light intensities that are orders of magnitude lower than other super-resolution methods. SIM is also far faster ...over cellular-sized fields of view. Li
et al.
used two approaches to improve the resolution of SIM to allow live cell imaging of dynamic cellular processes, including endocytosis and cytoskeleton remodeling. The contrast in performance between SIM and other techniques is due to a few key differences. Defining the practical resolution at the limited signal-to-noise ratios necessary for live cell imaging will require better imaging metrics.
Science
, this issue
10.1126/science.aab3500
Super-resolution imaging of fast dynamic processes in living cells is facilitated by improvements to structured illumination microscopy.
INTRODUCTION
Various methods of super-resolution (SR) fluorescence microscopy have the potential to follow the dynamic nanoscale interactions of specific macromolecular assemblies in living cells. However, this potential is often left unfulfilled, either owing to the method’s inability to follow these processes at the speeds dictated by nature or because they require intense light that can substantially perturb the very physiology one hopes to study. An exception is structured illumination microscopy (SIM), which can image live cells far faster and with orders of magnitude less light than required for other SR approaches. However, SIM’s resolution is usually limited to only a twofold gain beyond conventional optical microscopes, or ~100 nm with visible light.
RATIONALE
We endeavored to find ways to extend SIM to the sub-100-nm regime while retaining, to the greatest extent possible, the advantages that make it the preferred SR method for live-cell imaging. Our first solution used an ultrahigh numerical aperture (NA) lens and total internal reflection fluorescence (TIRF) to achieve 84-nm resolution at subsecond acquisition speeds over hundreds of time points in multiple colors near the basal plasma membrane. Our second exploited the spatially patterned activation of a recently developed, reversibly photoswitchable fluorescent protein to reach 45- to 62-nm resolution, also at subsecond acquisition, over ∼10 to 40 time points.
RESULTS
We used high-NA TIRF-SIM to image the dynamic associations of cortical filamentous actin with myosin IIA, paxillin, or clathrin, as well as paxillin with vinculin and clathrin with transferrin receptors. Thanks to the combination of high spatial and temporal resolution, we were able to measure the sizes of individual clathrin-coated pits through their initiation, growth, and internalization. We were also able to relate pit size to lifetime, identify and characterize localized hot spots of pit generation, and describe the interaction of actin with clathrin and its role in accelerating endocytosis. With nonlinear SIM by use of patterned activation (PA NL-SIM), we monitored the remodeling of the actin cytoskeleton and the dynamics of caveolae at the cell surface. By combining TIRF-SIM and PA NL-SIM for two-color imaging, we followed the dynamic association of actin with α-actinin in expanding filopodia and membrane ruffles and characterized shape changes in and the transport of early endosomes. Last, by combining PA NL-SIM with lattice light sheet microscopy, we observed, in three dimensions and across the entire volume of whole cells, the dynamics of the actin cytoskeleton, the fusion and fission of mitochondria, and the trafficking of vesicles to and from the Golgi apparatus, each at axial resolution fivefold better than that of conventional widefield microscopy.
In addition, through direct experimental comparisons, we demonstrated that the resolution for our methods is comparable with or better than other SR approaches yet allowed us to image at far higher speeds, and for far longer durations. To understand why this is so, we developed a detailed theoretical model showing that our methods transmit the information encoded in spatial frequencies beyond the diffraction limit with much greater strength than do other alternatives and hence require far fewer photons emitted from the specimen, using far less intense light.
CONCLUSION
High-NA TIRF-SIM and PA NL-SIM fill an unmet need for minimally invasive tools to image live cells in the gap between the 100-nm resolution traditionally associated with SIM and the sub-60-nm regime of protein-specific structural imaging served by single-molecule localization microscopy.
Two approaches for improved live-cell imaging at sub-100-nm resolution.
(
Left
) Association of cortical actin (purple) with clathrin-coated pits (green), the latter seen as rings (
inset
) at 84-nm resolution via a combination of total internal reflection fluorescence and structured illumination microscopy at ultrahigh numerical aperture (high-NA TIRF-SIM). (
Right
) Progression of resolution improvement across the actin cytoskeleton of a COS-7 cell, from conventional, diffraction-limited TIRF (220-nm resolution), to TIRF-SIM (97-nm resolution), and nonlinear SIM based on the patterned activation of a reversibly photoswitchable fluorescent protein (PA NL-SIM, 62 nm resolution). (Left and right represent single frames from time-lapse movies over 91 and 30 frames, respectively. Scale bars, 2 μm (left); 3 μm (right).
Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.
CLATHRIN Kirchhausen, Tomas
Annual review of biochemistry,
01/2000, Letnik:
69, Številka:
1
Journal Article
Recenzirano
Clathrin was discovered nearly 25 years ago. Since then, a large number of
other proteins that participate in the process by which clathrin-coated
vesicles retrieve synaptic membranes or take up ...endocytic receptors have been
identified. The functional relationships among these disparate components
remain, in many cases, obscure. High-resolution structures of parts of
clathrin, determined by X-ray crystallography, and lower-resolution images of
assembled coats, determined by electron cryomicroscopy, now provide the
information necessary to integrate various lines of evidence and to design
experiments that test specific mechanistic notions. This review summarizes and
illustrates the recent structural results and outlines what is known about
coated-vesicle assembly in the context of this information.
Celotno besedilo
Dostopno za:
CMK, DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The notion that AP-2 clathrin adaptor is an essential component of an endocytic clathrin coat appears to conflict with recent observations that substantial AP-2 depletion, using RNA interference with ...synthesis of AP-2 subunits, fails to block uptake of certain ligands known to internalize through a clathrin-based pathway.
We report here the use of in vivo imaging data obtained by spinning-disk confocal microscopy to study the formation of clathrin-coated structures at the plasma membranes of BSC1 and HeLa cells depleted by RNAi of the clathrin adaptor, AP-2. Very few clathrin coats continue to assemble after AP-2 knockdown. Moreover, there is a total absence of clathrin-containing structures completely lacking AP-2 while all the remaining coats still contain a small amount of AP-2. These observations suggest that AP-2 is essential for endocytic coated-pit and coated-vesicle formation. We also find that AP-2 knockdown strongly inhibits light-density lipoprotein (LDL) receptor-mediated endocytosis, as long as cells are maintained in complete serum and at 37 degrees C. If cells are first incubated with LDL at 4 degrees C, followed by warming, there is little or no decrease in LDL uptake with respect to control cells. LDL uptake at 37 degrees C is also not affected in AP-2 depleted cells first deprived of LDL by incubation with either serum-starved or LDL-starved cells for 24 hr. The LDL-deprived cells display a significant increase in endocytic structures enriched on deeply invaginated tubes that contain LDL and we suggest that under this condition of stress, LDL might enter through this alternative pathway.
These results suggest that AP-2 is essential for endocytic clathrin coated-pit and coated-vesicle formation. They also indicate that under normal conditions, functional endocytic clathrin coated pits are required for LDL internalization. We also show that under certain conditions of stress, cells can upregulate alternative endocytic structures with the potential to provide compensatory trafficking pathways.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The ESCRT machinery mediates reverse membrane scission. By quantitative fluorescence lattice light-sheet microscopy, we have shown that ESCRT-III subunits polymerize rapidly on yeast endosomes, ...together with the recruitment of at least two Vps4 hexamers. During their 3-45 s lifetimes, the ESCRT-III assemblies accumulated 75-200 Snf7 and 15-50 Vps24 molecules. Productive budding events required at least two additional Vps4 hexamers. Membrane budding was associated with continuous, stochastic exchange of Vps4 and ESCRT-III components, rather than steady growth of fixed assemblies, and depended on Vps4 ATPase activity. An all-or-none step led to final release of ESCRT-III and Vps4. Tomographic electron microscopy demonstrated that acute disruption of Vps4 recruitment stalled membrane budding. We propose a model in which multiple Vps4 hexamers (four or more) draw together several ESCRT-III filaments. This process induces cargo crowding and inward membrane buckling, followed by constriction of the nascent bud neck and ultimately ILV generation by vesicle fission.
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