Biology subjects

Reck-Peterson, S. L.

Publications and source records attributed to Reck-Peterson, S. L..

26 records · Page 2Linked to original sources

bioRxiv2021

Optimizing microtubule arrangements for rapid cargo capture

Cellular functions such as autophagy, cell signaling and vesicular trafficking involve the retrograde transport of motor-driven cargo along microtubules. Typically, newly formed cargo engages in slow diffusive movement from its point of origin before attaching to a microtubule. In some cell types, cargo destined for delivery to the perinuclear region relies on capture at dynein-enriched loading zones located near microtubule plus-ends. Such systems include extended cell regions of neurites and fungal hyphae, where the efficiency of the initial diffusive loading process depends on the axial distribution of microtubule plus-ends relative to the initial cargo position. We use analytic mean first passage time calculations and numerical simulations to model diffusive capture processes in tubular cells, exploring how the spatial arrangement of microtubule plus-ends affects the efficiency of retrograde cargo transport. Our model delineates the key features of optimal microtubule ar-rangements that minimize mean cargo capture times. Namely, we show that configurations with a single long microtubule and broad distribution of additional microtubule plus-ends allow for efficient capture in a variety of different scenarios for retrograde transport. Live-cell imaging of microtubule plus-ends in Aspergillus nidulans hyphae indicates that their distributions exhibit these optimal qualitative features. Our results highlight important coupling effects between microtubule length distribution and retrograde cargo transport, providing guiding principles for the spatial arrangement of microtubules within tubular cell regions.

biophysics↗

bioRxiv2020

Regulation of peroxisome and lipid droplet hitchhiking by PxdA and the DipA phosphatase

In canonical microtubule-based transport, adaptor proteins link cargos to the molecular motors dynein and kinesin. Recently, an alternative mode of transport known as hitchhiking was discovered, in which a cargo achieves motility by hitching a ride on an already-motile cargo, rather than attaching to a motor protein. Hitchhiking has been best-studied in two filamentous fungi, Aspergillus nidulans and Ustilago maydis. In U. maydis, ribonucleoprotein complexes, peroxisomes, lipid droplets, and endoplasmic reticulum all hitchhike on early endosomes. In A. nidulans, peroxisomes hitchhike using a putative molecular linker, PxdA, that associates with early endosomes. However, whether other organelles use PxdA to hitchhike on early endosomes is unclear, as are the molecular mechanisms that regulate hitchhiking in A. nidulans. Here we find that the proper distribution of lipid droplets, mitochondria and autophagosomes do not require PxdA, suggesting that PxdA is a molecular linker specific to peroxisomes. We also identify two new pxdA alleles, including a point mutation (R2044P) that disrupts PxdAs ability to associate with early endosomes and reduces peroxisome movement. Finally, we identify a novel regulator of peroxisome hitchhiking, the phosphatase DipA. DipA co-localizes with early endosomes and its early endosome-association relies on PxdA.

cell biology↗

bioRxiv2020

Parkinson's Disease-linked LRRK2 structure and model for microtubule interaction

Leucine Rich Repeat Kinase 2 (LRRK2) is the most commonly mutated gene in familial Parkinsons disease. LRRK2 is proposed to function in membrane trafficking and co-localizes with microtubules. We report the 3.5[A] structure of the catalytic half of LRRK2, and an atomic model of microtubule-associated LRRK2 built using a reported 14[A] cryo-electron tomography in situ structure. We propose that the conformation of LRRK2s kinase domain regulates its microtubule interaction, with a closed conformation favoring binding. We show that the catalytic half of LRRK2 is sufficient for microtubule binding and blocks the motility of the microtubule-based motors kinesin and dynein in vitro. Kinase inhibitors that stabilize an open conformation relieve this interference and reduce LRRK2 filament formation in cells, while those that stabilize a closed conformation do not. Our findings suggest that LRRK2 is a roadblock for microtubule-based motors and have implications for the design of therapeutic LRRK2 kinase inhibitors.

biophysics↗

bioRxiv2019

Hitching a Ride: Mechanics of Organelle Transport Through Linker-Mediated Hitchhiking

In contrast to the canonical picture of transport by direct attachment to motor proteins, recent evidence shows that a number of intracellular cargos navigate the cytoplasm by hitchhiking on motor-driven carrier organelles. We describe a quantitative model of intracellular cargo transport via hitchhiking, examining the efficiency of hitchhiking initiation as a function of geometric and mechanical parameters. We focus specifically on the parameter regime relevant to the hitchhiking motion of peroxisome organelles in fungal hyphae. Our work predicts the dependence of transport initiation rates on the distribution of cytoskeletal tracks and carrier organelles, as well as the number, length and flexibility of the linker proteins that mediate contact between the carrier and the hitchhiking cargo. Furthermore, we demonstrate that attaching organelles to microtubules can result in a substantial enhancement of the hitchhiking initiation rate in tubular geometries such as those found in fungal hyphae. This enhancement is expected to increase the overall transport rate of hitchhiking organelles, and lead to greater efficiency in organelle dispersion. Our results leverage a quantitative physical model to highlight the importance of organelle encounter dynamics in non-canonical intracellular transport.\n\nSIGNIFICANCEA variety of cellular components are transported via hitchhiking by attaching to other motile organelles. Defects in the molecular machinery responsible for organelle hitchhiking may be linked with neurodegenerative disorders. To date, no comprehensive physical models of this non-canonical mode of transport have been developed. In particular, the connection between molecular- and organelle-scale properties of hitchhiking components and their effect on cellular-scale transport has remained unclear. Here, we investigate the mechanics of hitchhiking initiation and explore organelle interactions that can modulate the efficiency of this process.

biophysics↗

bioRxiv2019

Lis1 promotes the formation of maximally activated cytoplasmic dynein-1 complexes

Cytoplasmic dynein-1 is a molecular motor that drives nearly all minus-end-directed microtubule-based transport in human cells, performing functions ranging from retrograde axonal transport to mitotic spindle assembly1,2. Activated dynein complexes consist of one or two dynein dimers, the dynactin complex, and an \"activating adaptor\", with maximal velocity seen with two dimers present (Fig. 1a)3-6. Little is known about how this massive [~]4MDa complex is assembled. Using purified recombinant human proteins, we uncovered a novel role for the dynein-binding protein, Lis1, in the formation of fully activated dynein complexes containing two dynein dimers. Lis1 is required for maximal velocity of complexes activated by proteins representing three different families of activating adaptors: BicD2, Hook3, and Ninl. Once activated dynein complexes have formed, they do not require the presence of Lis1 for sustained maximal velocity. Using cryo-electron microscopy we show that human Lis1 binds to dynein at two sites on dyneins motor domain, similar to yeast dynein7. We propose that the ability of Lis1 to bind at these sites may function in multiple stages of assembling the motile human dynein/ dynactin/ activating adaptor complex.\n\nO_FIG O_LINKSMALLFIG WIDTH=123 HEIGHT=200 SRC=\"FIGDIR/small/683052v1_fig1.gif\" ALT=\"Figure 1\">\nView larger version (37K):\norg.highwire.dtl.DTLVardef@1acc26corg.highwire.dtl.DTLVardef@1107393org.highwire.dtl.DTLVardef@113eab4org.highwire.dtl.DTLVardef@95f6f4_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO Lis1 increases microtubule binding and the velocity of activated dynein complexes. a. Schematic of the current model for dynein activation. Dynein is autoinihibited in the Phi conformation, opens and then adopts a parallel conformation that is seen in the activated dynein complex, which includes dynactin and an activating adaptor. Maximally activated dynein contains two dynein dimers (dynein A and B, far right) b. Schematic of the dynein motor domain with AAA+ ATPase domains colored in rainbow, highlighting the two Lis1 binding sites in yeast dynein, \"sitering\" at AAA3/4 and \"sitestalk\" on dyneins stalk, which leads to dyneins microtubule binding domain (MTBD). Lis1 is shown in orange to the right c. Schematic of the activating adaptor constructs used in this study. d. Binding density (mean {+/-} s.e.m. dynein) of dynein alone on microtubules in the absence (white circles) or presence (black circles) of 300 nM Lis1. Data was normalized to a density of 1.0 in the absence of Lis1. Statistical analysis was performed using a two-tailed unpaired t test; ****, p<0.0001; n = 12 replicates for each condition. e. Binding density (mean {+/-} s.e.m) of dynein/ dynactin/ activating adaptor complexes on microtubules in the absence (white circles) or presence (black circles) of 300 nM Lis1. The activating adaptors used are indicated. Data was normalized to a density of 1.0 in the absence of Lis1. Statistical analysis was performed using a two-tailed unpaired t test; ****, p<0.0001; n = 12 replicates for each condition. f. Velocity of dynein/ dynactin/ activating adaptor complexes in the absence (white circles) or presence (black circles) of 300 nM Lis1. The median and interquartile range are shown and the activating adaptors used are indicated. Statistical analysis was performed using a two-tailed Mann-Whitney test; ****, p<0.0001; ns, p=0.3498; n (individual single molecule events) = 506 (BicD2-S no Lis1), 569 (BicD2-S with Lis1), 496 (BicD2-L no Lis1), 505 (BicD2-L with Lis1), 454 (Hook3 no Lis1), 471 (Hook3 with Lis1), 490 (Ninl no Lis1), 582 (Ninl with Lis1). g. Percent processive runs (mean {+/-} s.e.m.) of dynein/ dynactin/ Hook3 complexes in a higher salt buffer (60 mM KOAc versus 30 mM KOAc in our standard motility buffer) in the absence (white circles) or presence (black circles) of 300 nM Lis1. Statistical analysis was performed using a one-way ANOVA and Tukeys multiple comparisons test; ****, p<0.0001; n = 3 replicates per condition. h. Schematic of the peroxisome relocation assay. i. Peroxisome velocity in human U2OS cells with scrambled or Lis1 siRNA knockdown. The median and interquartile range are shown. Statistical analysis was performed using a two-tailed unpaired t test; ***, p=0.0002; n (average peroxisome velocity per cell) = 30 per condition. More than 7 events were measured per cell.\n\nC_FIG

biochemistry↗

bioRxiv2018

HOOK3 is a scaffold for the opposite-polarity microtubule-based motors cytoplasmic dynein and KIF1C

The unidirectional and opposite-polarity microtubule-based motors, dynein and kinesin, drive long-distance intracellular cargo transport. Cellular observations support the existence of mechanisms to couple opposite polarity motors: in cells some cargos rapidly switch directions and kinesin motors can be used to localize dynein. We recently identified an interaction between the cytoplasmic dynein-1 activating adaptor HOOK3 and the kinesin-3 KIF1C. Here we show that KIF1C and dynein/dynactin can exist in a single complex scaffolded by HOOK3. Full-length HOOK3 binds to and activates dynein/dynactin motility. HOOK3 also binds to a short region in the \"tail\" of KIF1C, but unlike dynein/dynactin, this interaction does not affect the processive motility of KIF1C. HOOK3 scaffolding allows dynein to transport KIF1C towards the microtubule minus end, and KIF1C to transport dynein towards the microtubule plus end. We propose that linking dynein and kinesin motors by dynein activating adaptors may be a general mechanism to regulate bidirectional motility.

cell biology↗

bioRxiv2017

Lis1 has two opposing modes of regulating cytoplasmic dynein

Regulation is central to the functional versatility of cytoplasmic dynein, a motor involved in intracellular transport, cell division, and neurodevelopment. Previous work established that Lis1, a conserved and ubiquitous regulator of dynein, binds to its motor domain and induces a tight microtubule-binding state in dynein. The work we present here--a combination of biochemistry, single-molecule assays, cryo-electron microscopy and in vivo experiments--led to the surprising discovery that Lis1 has two opposing modes of regulating dynein, being capable of inducing both low and high affinity for the microtubule. We show that these opposing modes depend on the stoichiometry of Lis1 binding to dynein and that this stoichiometry is regulated by the nucleotide state of dyneins AAA3 domain. We present data on the in vitro and in vivo consequences of abolishing the novel Lis1-induced weak microtubule-binding state in dynein and propose a new model for the regulation of dynein by Lis1.

cell biology↗

bioRxiv2017

The Human Cytoplasmic Dynein Interactome Reveals Novel Activators Of Motility

In human cells, cytoplasmic dynein-1 is essential for long-distance transport of many cargos, including organelles, RNAs, proteins, and viruses, towards microtubule minus ends. To understand how a single motor achieves cargo specificity, we identified the human dynein interactome or \"transportome\" by attaching a promiscuous biotin ligase (\"BioID\") to seven components of the dynein machinery, including a subunit of the essential cofactor dynactin. This method reported spatial information about the large cytosolic dynein/dynactin complex in living cells. To achieve maximal motile activity and to bind its cargos, human dynein/dynactin requires \"activators\", of which only five have been described. We developed methods to identify new activators in our BioID data, and discovered that ninein and ninein-like are a new family of dynein activators. Analysis of the protein interactomes for six activators, including ninein and ninein-like, suggests that each dynein activator has multiple cargos.

cell biology↗