ESCRT-0 marks an APPL1-independent transit route for EGFR between the cell surface and the EEA1-positive early endosome.

Endosomal sorting complexes required for transport (ESCRT)-0 sorts ubiquitylated EGFR within the early endosome so that the receptor can be incorporated into intralumenal vesicles. An important question is whether ESCRT-0 acts solely upon EGFR that has already entered the vacuolar early endosome (characterised by the presence of EEA1) or engages EGFR within earlier compartments. Here, we employ a suite of software to determine the localisation of ESCRT-0 at subpixel resolution and to perform particle-based colocalisation analysis with other endocytic markers. We demonstrate that although some of the ESCRT-0 subunit Hrs (also known as HGS) colocalises with the vacuolar early endosome marker EEA1, most localises to a population of peripheral EEA1-negative endosomes that act as intermediates in transporting EGFR from the cell surface to more central early endosomes. The peripheral Hrs-labelled endosomes are distinct from APPL1-containing endosomes, but co-label with the novel endocytic adaptor SNX15. In contrast to ESCRT-0, ESCRT-I is recruited to EGF-containing endosomes at later times as they move to more a central position, whereas ESCRT-III is also recruited more gradually. RNA silencing experiments show that both ESCRT-0 and ESCRT-I are important for the transit of EGF to EEA1 endosomes.

The Charcot Marie Tooth disease protein LITAF is a zinc-binding monotopic membrane protein.

LITAF (LPS-induced TNF-activating factor) is an endosome-associated integral membrane protein important for multivesicular body sorting. Several mutations in LITAF cause autosomal-dominant Charcot Marie Tooth disease type 1C. These mutations map to a highly conserved C-terminal region, termed the LITAF domain, which includes a 22 residue hydrophobic sequence and flanking cysteine-rich regions that contain peptide motifs found in zinc fingers. Although the LITAF domain is thought to be responsible for membrane integration, the membrane topology of LITAF has not been established. Here, we have investigated whether LITAF is a tail-anchored (TA) membrane-spanning protein or monotopic membrane protein. When translated in vitro, LITAF integrates poorly into ER-derived microsomes compared with Sec61ß, a bona fide TA protein. Furthermore, introduction of N-linked glycosylation reporters shows that neither the N-terminal nor C-terminal domains of LITAF translocate into the ER lumen. Expression in cells of an LITAF construct containing C-terminal glycosylation sites confirms that LITAF is not a TA protein in cells. Finally, an immunofluorescence-based latency assay showed that both the N- and C-termini of LITAF are exposed to the cytoplasm. Recombinant LITAF contains 1 mol/mol zinc, while mutation of predicted zinc-binding residues disrupts LITAF membrane association. Hence, we conclude that LITAF is a monotopic membrane protein whose membrane integration is stabilised by a zinc finger. The related human protein, CDIP1 (cell death involved p53 target 1), displays identical membrane topology, suggesting that this mode of membrane integration is conserved in LITAF family proteins.

Multivesicular bodies: co-ordinated progression to maturity.

Multivesicular endosomes/bodies (MVBs) sort endocytosed proteins to different destinations. Many lysosomally directed membrane proteins are sorted onto intralumenal vesicles, whilst recycling proteins remain on the perimeter membrane from where they are removed via tubular extensions. MVBs move to the cell centre during this maturation process and, when all recycling proteins have been removed, fuse with lysosomes. Recent advances have identified endosomal-sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways in intralumenal vesicle formation and mechanisms for sorting recycling cargo into tubules. Cytoskeletal motors, through interactions with these machineries and by regulating MVB movement, help to co-ordinate events leading to a mature, fusion-competent MVB.

Leucine-rich repeat kinase 2 regulates autophagy through a calcium-dependent pathway involving NAADP.

Mutations in the leucine-rich repeat kinase-2 (LRRK2) gene cause late-onset Parkinson's disease, but its physiological function has remained largely unknown. Here we report that LRRK2 activates a calcium-dependent protein kinase kinase-ß (CaMKK-ß)/adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway which is followed by a persistent increase in autophagosome formation. Simultaneously, LRKR2 overexpression increases the levels of the autophagy receptor p62 in a protein synthesis-dependent manner, and decreases the number of acidic lysosomes. The LRRK2-mediated effects result in increased sensitivity of cells to stressors associated with abnormal protein degradation. These effects can be mimicked by the lysosomal Ca(2+)-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) and can be reverted by an NAADP receptor antagonist or expression of dominant-negative receptor constructs. Collectively, our data indicate a molecular mechanism for LRRK2 deregulation of autophagy and reveal previously unidentified therapeutic targets.

Dynein is required for receptor sorting and the morphogenesis of early endosomes.

The early endosome is organised into domains to ensure the separation of cargo. Activated mitogenic receptors, such as epidermal growth factor (EGF) receptor, are concentrated into vacuoles enriched for the small GTPase Rab5, which progressively exclude nutrient receptors, such as transferrin receptor, into neighbouring tubules. These vacuoles become enlarged, increase their content of intralumenal vesicles as EGF receptor is sorted from the limiting membrane, and eventually mature to late endosomes. Maturation is governed by the loss of Rab5 and is accompanied by the movement of endosomes along microtubules towards the cell centre. Here, we show that EGF relocates to the cell centre in a dynein-dependent fashion, concomitant with the sorting away of transferrin receptor, although it remains in Rab5-positive early endosomes. When dynein function is acutely disrupted, efficient recycling of transferrin from EGF-containing endosomes is retarded, loss of Rab5 is slowed and endosome enlargement is reduced.

Modes of correlated angular motion in live cells across three distinct time scales.

Particle tracking experiments with high speed digital microscopy yield the positions and trajectories of lipid droplets inside living cells. Angular correlation analysis shows that the lipid droplets have uncorrelated motion at short time scales (τ < 1 ms) followed by anti-persistent motion for lag times in the range of 1 ⩽ τ ⩽ 10 ms. The angular correlation at longer time scales, τ > 10 ms, becomes persistent, indicating directed movement. The motion at all time scales is associated with the lipid droplets being tethered to and driven along the microtubule network. The point at which the angular correlation changes from anti-persistent to persistent motion corresponds to the cross over between sub-diffusive and super diffusive motion, as observed by mean square displacement analysis. Correct analysis of the angular correlations of the detector noise is found to be crucial in modelling the observed phenomena.

The meiotic LINC complex component KASH5 is an activating adaptor for cytoplasmic dynein.

Cytoplasmic dynein-driven movement of chromosomes during prophase I of mammalian meiosis is essential for synapsis and genetic exchange. Dynein connects to chromosome telomeres via KASH5 and SUN1 or SUN2, which together span the nuclear envelope. Here, we show that KASH5 promotes dynein motility in vitro, and cytosolic KASH5 inhibits dynein's interphase functions. KASH5 interacts with a dynein light intermediate chain (DYNC1LI1 or DYNC1LI2) via a conserved helix in the LIC C-terminal, and this region is also needed for dynein's recruitment to other cellular membranes. KASH5's N-terminal EF-hands are essential as the interaction with dynein is disrupted by mutation of key calcium-binding residues, although it is not regulated by cellular calcium levels. Dynein can be recruited to KASH5 at the nuclear envelope independently of dynactin, while LIS1 is essential for dynactin incorporation into the KASH5-dynein complex. Altogether, we show that the transmembrane protein KASH5 is an activating adaptor for dynein and shed light on the hierarchy of assembly of KASH5-dynein-dynactin complexes.

Functional interplay between LIS1, NDE1 and NDEL1 in dynein-dependent organelle positioning.

LIS1, NDE1 and NDEL1 modulate cytoplasmic dynein function in several cellular contexts. However, evidence that they regulate dynein-dependent organelle positioning is limited. Here, we show that depletion of NDE1 or NDEL1 alone profoundly affected the organisation of the Golgi complex but did not cause it to disperse, and slightly affected the position of endocytic compartments. However, striking dispersal of organelles was observed when both NDE1 and NDEL1 were depleted. A substantial portion of NDE1 and NDEL1 is membrane associated, and depletion of these proteins led to complete loss of dynein from membranes. Knockdown of LIS1 also caused the Golgi complex to fragment and disperse throughout the cell, and caused endocytic compartments to relocalise to the periphery. Depletion of LIS1, which is primarily cytosolic, led to partial loss of membrane-associated dynein, without affecting NDE1 and NDEL1. These data suggest that NDE1 and NDEL1 act upstream of LIS1 in dynein recruitment, and/or activation, on the membrane. Consistent with this hypothesis, expression of exogenous NDE1 or NDEL1 rescued the effects of LIS1 depletion on Golgi organisation, whereas LIS1 was only partially effective at rescuing the loss of NDE1 and NDEL1.

The first passage probability of intracellular particle trafficking.

The first passage probability (FPP), of trafficked intracellular particles reaching a displacement L, in a given time t or inverse velocity S = t/L, can be calculated robustly from measured particle tracks. The FPP gives a measure of particle movement in which different types of motion, e.g. diffusion, ballistic motion, and transient run-rest motion, can readily be distinguished in a single graph, and compared with mathematical models. The FPP is attractive in that it offers a means of reducing the data in the measured tracks, without making assumptions about the mechanism of motion. For example, it does not employ smoothing, segmentation or arbitrary thresholds to discriminate between different types of motion in a particle track. In contrast to conventional mean square displacement analysis, FPP is sensitive to a small population of trafficked particles that move long distances (> or = 5 microm), which are thought to be crucial for efficient long range signaling in theories of network dynamics. Taking experimental data from tracked endocytic vesicles, and calculating the FPP, we see how molecular treatments affect the trafficking. We show the FPP can quantify complicated movement which is neither completely random nor completely deterministic, making it highly applicable to trafficked particles in cell biology.

Membrane trafficking in health and disease.

Membrane trafficking pathways are essential for the viability and growth of cells, and play a major role in the interaction of cells with their environment. In this At a Glance article and accompanying poster, we outline the major cellular trafficking pathways and discuss how defects in the function of the molecular machinery that mediates this transport lead to various diseases in humans. We also briefly discuss possible therapeutic approaches that may be used in the future treatment of trafficking-based disorders.

Caspase-mediated cleavage of the stacking protein GRASP65 is required for Golgi fragmentation during apoptosis.

The mammalian Golgi complex is comprised of a ribbon of stacked cisternal membranes often located in the pericentriolar region of the cell. Here, we report that during apoptosis the Golgi ribbon is fragmented into dispersed clusters of tubulo-vesicular membranes. We have found that fragmentation is caspase dependent and identified GRASP65 (Golgi reassembly and stacking protein of 65 kD) as a novel caspase substrate. GRASP65 is cleaved specifically by caspase-3 at conserved sites in its membrane distal COOH terminus at an early stage of the execution phase. Expression of a caspase-resistant form of GRASP65 partially preserved cisternal stacking and inhibited breakdown of the Golgi ribbon in apoptotic cells. Our results suggest that GRASP65 is an important structural component required for maintenance of Golgi apparatus integrity.

The flexibility and dynamics of the tubules in the endoplasmic reticulum.

The endoplasmic reticulum (ER) is a single organelle in eukaryotic cells that extends throughout the cell and is involved in a large number of cellular functions. Using a combination of fixed and live cells (human MRC5 lung cells) in diffraction limited and super-resolved fluorescence microscopy (STORM) experiments, we determined that the average persistence length of the ER tubules was 3.03 ± 0.24 µm. Removing the branched network junctions from the analysis caused a slight increase in the average persistence length to 4.71 ± 0.14 µm, and provides the tubule's persistence length with a moderate length scale dependence. The average radius of the tubules was 44.1 ± 3.2 nm. The bending rigidity of the ER tubule membranes was found to be 10.9 ± 1.2 kT (17.0 ± 1.3 kT without branch points). We investigated the dynamic behaviour of ER tubules in live cells, and found that the ER tubules behaved like semi-flexible fibres under tension. The majority of the ER tubules experienced equilibrium transverse fluctuations under tension, whereas a minority number of them had active super-diffusive motions driven by motor proteins. Cells thus actively modulate the dynamics of the ER in a well-defined manner, which is expected in turn to impact on its many functions.

Dynein light intermediate chains maintain spindle bipolarity by functioning in centriole cohesion.

Cytoplasmic dynein 1 (dynein) is a minus end-directed microtubule motor protein with many cellular functions, including during cell division. The role of the light intermediate chains (LICs; DYNC1LI1 and 2) within the complex is poorly understood. In this paper, we have used small interfering RNAs or morpholino oligonucleotides to deplete the LICs in human cell lines and Xenopus laevis early embryos to dissect the LICs' role in cell division. We show that although dynein lacking LICs drives microtubule gliding at normal rates, the LICs are required for the formation and maintenance of a bipolar spindle. Multipolar spindles with poles that contain single centrioles were formed in cells lacking LICs, indicating that they are needed for maintaining centrosome integrity. The formation of multipolar spindles via centrosome splitting after LIC depletion could be rescued by inhibiting Eg5. This suggests a novel role for the dynein complex, counteracted by Eg5, in the maintenance of centriole cohesion during mitosis.

First-passage-probability analysis of active transport in live cells.

The first-passage-probability can be used as an unbiased method for determining the phases of motion of individual organelles within live cells. Using high speed microscopy, we observe individual lipid droplet tracks and analyze the motor protein driven motion. At short passage lengths (<10(-2)µm), a log-normal distribution in the first-passage-probability as a function of time is observed, which switches to a Gaussian distribution at longer passages due to the running motion of the motor proteins. The mean first-passage times () as a function of the passage length (L), averaged over a number of runs for a single lipid droplet, follow a power law distribution ~L(α), α>2, at short times due to a passive subdiffusive process. This changes to another power law at long times where 1<α<2, corresponding to sub-ballistic superdiffusive motion, an active process. Subdiffusive passive mean square displacements are observed as a function of time, ~t(ß), where 0<ß<1 at short times again crossing over to an active sub-ballistic superdiffusive result 1<ß<2 at longer times. Consecutive runs of the lipid droplets add additional independent Gaussian peaks to a cumulative first-passage-probability distribution indicating that the speeds of sequential phases of motion are independent and biochemically well regulated. As a result we propose a model for motor driven lipid droplets that exhibits a sequential run behavior with occasional pauses.

Roles of dynein and dynactin in early endosome dynamics revealed using automated tracking and global analysis.

Microtubule-dependent movement is crucial for the spatial organization of endosomes in most eukaryotes, but as yet there has been no systematic analysis of how a particular microtubule motor contributes to early endosome dynamics. Here we tracked early endosomes labeled with GFP-Rab5 on the nanometer scale, and combined this with global, first passage probability (FPP) analysis to provide an unbiased description of how the minus-end microtubule motor, cytoplasmic dynein, supports endosome motility. Dynein contributes to short-range endosome movement, but in particular drives 85-98% of long, inward translocations. For these, it requires an intact dynactin complex to allow membrane-bound p150(Glued) to activate dynein, since p50 over-expression, which disrupts the dynactin complex, inhibits inward movement even though dynein and p150(Glued) remain membrane-bound. Long dynein-dependent movements occur via bursts at up to ∼8 µms(-1) that are linked by changes in rate or pauses. These peak speeds during rapid inward endosome movement are still seen when cellular dynein levels are 50-fold reduced by RNAi knock-down of dynein heavy chain, while the number of movements is reduced 5-fold. Altogether, these findings identify how dynein helps define the dynamics of early endosomes.

A role for soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex dimerization during neurosecretion.

The interactions underlying the cooperativity of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes during neurotransmission are not known. Here, we provide a molecular characterization of a dimer formed between the cytoplasmic portions of neuronal SNARE complexes. Dimerization generates a two-winged structure in which the C termini of cytosolic SNARE complexes are in apposition, and it involves residues from the vesicle-associated SNARE synaptobrevin 2 that lie close to the cytosol-membrane interface within the full-length protein. Mutation of these residues reduces stability of dimers formed between SNARE complexes, without affecting the stability of each individual SNARE complex. These mutations also cause a corresponding decrease in the ability of botulinum toxin-resistant synaptobrevin 2 to rescue regulated exocytosis in toxin-treated neuroendocrine cells. Moreover, such synaptobrevin 2 mutants give rise to a dominant-negative inhibition of exocytosis. These data are consistent with an important role for SNARE complex dimers in neurosecretion.

Active relocation of chromatin and endoplasmic reticulum into blebs in late apoptotic cells.

Plasma membrane blebbing is a defining characteristic of apoptosis, but its significance is not understood. Using live-cell imaging we have identified two phases of apoptotic blebbing. The early phase is restricted to adherent cells, and is prevented by the Rho-activated kinase inhibitor Y27632. The late phase is partially resistant to Y27632, and generates morphologically distinct membrane protrusions that are likely precursors to apoptotic bodies. Late blebbing is observed in all apoptotic cells tested. It occurs at a fixed period before phosphatidyl serine exposure, indicating that it is a universal and important feature of apoptosis. Late blebs contain a cortical layer of endoplasmic reticulum that often surrounds condensed chromatin, while other organelles are excluded. The appearance in some apoptotic cells of partially formed sheets of endoplasmic reticulum suggest that these cortical layers are newly formed by the remodelling of the endoplasmic reticulum of interphase cells. Formation of endoplasmic reticulum and chromatin-containing blebs requires both actin and microtubules, and is prevented by the caspase-6 inhibitor zVEID.fmk.

Depletion of TSG101 forms a mammalian "Class E" compartment: a multicisternal early endosome with multiple sorting defects.

The early endosome comprises morphologically distinct regions specialised in sorting cargo receptors. A central question is whether receptors move through a predetermined structural pathway, or whether cargo selection contributes to the generation of endosome morphology and membrane flux. Here, we show that depletion of tumour susceptibility gene 101 impairs the selection of epidermal growth factor receptor away from recycling receptors within the limiting membrane of the early endosome. Consequently, epidermal growth factor receptor sorting to internal vesicles of the multivesicular body and cargo recycling to the cell surface or Golgi complex are inhibited. These defects are accompanied by disruption of bulk flow transport to the lysosome and profound structural rearrangement of the early endosome. The pattern of tubular and vacuolar domains is replaced by enlarged vacuoles, many of which are folded into multicisternal structures resembling the "Class E" compartments that define several Saccharomyces cerevisiae vacuolar protein sorting mutants. The cisternae are interleaved by a fine matrix but lack other surface elaborations, most notably clathrin.

p97, a protein coping with multiple identities.

A topic that is keeping cell biologists across several fields occupied is how the AAA ATPase p97 can have so many apparently unrelated functions. A recent model that proposed sets of adaptors for p97 selected according to the type of p97 activity seemed to afford a simple solution. For example, one known adaptor, the Ufd1-Npl4 complex, has been implicated in ubiquitin-dependent proteolysis whereas another, p47, is an essential co-factor for membrane fusion. However, further investigation has revealed that the situation is more complicated. Both Ufd1-Npl4 and p47 adaptors bind ubiquitin, and so their activities may be more closely related than first thought. A role for ubiquitin in p97-dependent membrane fusion is a particularly surprising development with no obvious explanation. However, some clues may be found from looking at the role of ubiquitin and the AAA ATPase Vps4 during sorting on the endocytic pathway.

Caspase-mediated cleavage of syntaxin 5 and giantin accompanies inhibition of secretory traffic during apoptosis.

We report the caspase-dependent cleavage of two Golgi-associated transport factors during apoptosis. The tethering factor giantin is rapidly cleaved both in vitro and in vivo at a conserved site, to generate a stable membrane-anchored domain and a soluble domain that is subject to further caspase-dependent cleavage. The t-SNARE syntaxin 5 is also cleaved rapidly, resulting in the separation of the catalytic membrane-proximal domain from an N-terminal regulatory domain. Cleavage of giantin and syntaxin 5 is accompanied by a cessation of vesicular transport between the ER and the Golgi complex, which first manifests itself as a block in ER exit. The contribution that such an inhibition of trafficking may make towards the generation of an apoptotic phenotype is discussed.