#intracellular transport

Basics of Cell Biology

Which cellular structure plays a role in intracellular transport and is part of the cytoskeleton?

Cytoskeleton provides scaffold and track system built from protein polymers regulated by nucleation factors, GTPases, and post-translational modifications. Actin filaments, 7 nanometer diameter polarized structures polymerizing from ATP-G-actin mediated by Arp2/3 complex branching at 70 degrees and formins nucleating straight cables for filopodia, associate with myosin II for contractility at cortex and myosin V for short-range vesicle transport near cell periphery, driving lamellipodia extension and endocytic invagination. Microtubules, 25 nanometer hollow tubes of alpha-beta tubulin dimers with GTP hydrolysis driving dynamic instability with growth and catastrophe phases, originate at microtubule organizing center centrosome and serve as long-distance highways for kinesin anterograde and dynein retrograde motors carrying organelles, mRNA granules, and lysosomes, forming mitotic spindle via kinetochore attachment through Ndc80 complex ensuring chromosome segregation. Intermediate filaments, 10 nanometer non-polar ropes including keratin in epithelia and vimentin in mesenchyme anchored by plectin, confer tensile strength protecting nucleus. All three together constitute cytoskeletal network supporting transport, mechanical integrity, and motility essential for cell physiology.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 16: Cytoskeleton and Intracellular Transport.

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Cell Organelles

Early endosomes function in:

Endocytic trafficking relies on progressive maturation and sorting of internalized cargo to maintain membrane homeostasis and signaling fidelity. Early endosomes, also called sorting endosomes, receive clathrin-coated and non-clathrin vesicles from the plasma membrane containing receptors such as transferrin receptor, EGF receptor, ligands, low-density lipoprotein, and extracellular fluid. Their pleomorphic tubular-vesicular morphology and mildly acidic pH around 6.0 to 6.2, maintained by vacuolar ATPase delivering protons, facilitate dissociation of many ligands from receptors while preserving receptor structure. Rab5 GTPase in active GTP-bound state and its effectors EEA1 tether, phosphatidylinositol-3-phosphate lipid platform, and SNARE syntaxin 13 coordinate homotypic fusion and recruitment of sorting machineries. Geometrically distinct sorting domains segregate cargo for divergent fates: receptors like transferrin receptor recycle to plasma membrane via Rab4 fast loop and Rab11 slow perinuclear recycling routes, whereas ubiquitinated growth factor receptors are captured by ESCRT-0, I, II, III complexes into intraluminal vesicles directing toward multivesicular bodies for degradation. Lipids, nutrients and signaling molecules are similarly triaged. This organizing function prevents unnecessary degradation, sustains surface receptor homeostasis while initiating downstream MAPK signaling from endosomal platforms.

Ref: Alberts et al., Molecular Biology of the Cell, 6th ed., Chapter 13: Intracellular Vesicular Traffic – Early Endosomes and Sorting.

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Cytoskeleton

What is the function of the dynactin complex in dynein activity?

Although dynein can bind microtubules and hydrolyze ATP alone in vitro motility assays in vivo most physiological functions require dynactin 1 MDa multiprotein complex discovered as activator of vesicle transport from squid axoplasm. Structure comprises Arp1 actin related protein filament 8 copies forming short filament 37 nm rod barbed end capped by CapZ pointed end by Arp11 p62 p25 p27 complex shoulder arm containing p150Glued DCTN1 with N terminal CAP Gly plus basic microtubule binding domains and long coiled coil dimer interacting with dynein intermediate chain via CC1 box. Dynactin performs dual roles: binding diverse cargo adaptors BicD2 Hook3 Spindly Ninein that select vesicles nuclei kinetochores via coiled coil cargo binding domains and tethering dynein to microtubules via p150Glued MTBD increasing processivity from limited hundred nanometer runs to several microns run length up to 10 microns. Cargo binding relieves dynein autoinhibition converting weak diffusive complex into high force super complex containing two dynein dimers for faster movement. Therefore dynactin links dynein to cargo and enhances processivity not inhibiting.

Ref: Schroer, Annu Rev Cell Dev Biol 2004 – Dynactin function linking dynein to cargo and enhancing processivity.

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Cytoskeleton

What regulates kinesin-1 activation?

Kinesin-1 regulation avoids gratuitous ATP consumption and traffic jams by autoinhibition mechanism evolutionarily conserved ensuring motor only active when bound to cargo. In cargo free state heavy chain tail IAK motif folds back contacting motor head switch regions blocking microtubule stimulated ADP release maintaining ATPase low about 10 fold inhibition. Light chains also contribute to folded globular conformation compact. Cargo adaptors such as JNK interacting proteins JIP1-3 FEZ1 Milton TRAK bind light chains or tail sterically unfolding molecule to extended active state increasing microtubule affinity 20 fold and ATPase rate. ATP hydrolysis itself occurs in motor domain P loop driving stepping not in stalk domain which serves purely structural coiled coil dimerization role linking heads. Thus activation triggered by cargo binding induced unfolding not stalk ATP hydrolysis. ATP hydrolysis at motor provides energy for movement. This regulatory paradigm ensures motor travels only when loaded linking ATP cycles to cargo availability for processive anterograde movement failure leads to organelle mislocalization neurodegeneration and developmental defects.

Ref: Verhey & Hammond, Nat Rev Mol Cell Biol 2009 – Kinesin-1 activation by cargo binding unfolding mechanism.

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Cytoskeleton

What is the approximate step size of Myosin V during intracellular transport?

Myosin V stride size uniquely adapted to actin filament helical symmetry enabling straight long distance walks without spiraling around filament axis. The molecule possesses six IQ motifs per heavy chain creating extended lever about 24 nm much longer than myosin II lever. During processive movement heads alternate hand over hand trailing head detaches after ATP binding swings forward as lever rotates about 100 degrees. Actin filament repeats every 13 monomers corresponding to 36 nm axial distance where binding site orientation repeats facing same direction. Center of mass therefore advances 36 nm per ATP hydrolysis cycle matching repeat. Detailed single molecule tracking using optical trap and fluorescence showed individual head moves 72 nm per step while other remains attached ensuring dimer spans two helical repeats. This large step allows melanosomes vacuoles and mRNA to be carried efficiently through cortical actin and explains why artificial short lever mutants show reduced processivity and frequent detachment. Coordination via internal strain dependent gating prevents both heads detaching simultaneously maintaining high duty ratio.

Ref: Mehta et al., Nature 1999; Purcell et al., PNAS 2005 – Myosin V 36 nm center and 72 nm head step mechanism.

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Cytoskeleton

What is the role of Myosin V in intracellular transport?

Myosin V is prototypical class V unconventional myosin that operates on actin filaments not microtubules serving local delivery after long range microtubule transport. The molecule is dimeric with two motor heads each containing six calmodulin or essential light chain IQ motifs forming a 24 nm lever arm, a coiled coil stalk and a globular tail domain that binds cargo adaptors such as Rab11, Rab27, melanophilin and Myo4p binding proteins. It moves processively hand over hand toward barbed plus end near plasma membrane hydrolyzing one ATP per step with coordinated gating that prevents simultaneous detachment. The motor center of mass advances 36 nm per ATP matching actin helical repeat of 13 monomers while each head swings about 72 nm. This architecture allows organelles, secretory vesicles, endoplasmic reticulum tubules, mRNA granules and melanosomes to traverse dense cortical actin networks where kinesin and dynein cannot operate. Regulation involves cargo binding relieving autoinhibition, calcium calmodulin influencing lever stiffness and coincidence detection with Rab GTPases ensuring correct delivery and recycling.

Ref: Vale, Cell 2003; Hammer & Sellers, Nat Rev Mol Cell Biol 2012 – Myosin V processive transport on actin filaments.

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Cytoskeleton

Which myosin type is responsible for intracellular cargo transport?

Intracellular cargo trafficking covers both microtubule and actin routes. Long range 10 to 100 micron interphase transport uses kinesin dynein on microtubules, while short range local transport within cortical actin rich zones 1 to 10 micron uses myosin. Processive myosin capable of multiple steps without dissociation required for efficient transport. Myosin V class features high duty ratio spending about 70 percent cycle strongly attached, dimeric coiled coil tail, six IQ motifs with calmodulin light chains per head forming long 24 nanometer lever arm achieving 36 nanometer step matching helical repeat of actin, hand over hand walking mechanism with gating coordinated by intramolecular strain. Transport cargos include melanosomes via interaction with melanophilin Rab27a in melanocytes, synaptic vesicles, endoplasmic reticulum and mRNA in budding yeast via Myo2 Myo4. Myosin I monomeric membrane tether, myosin II bipolar contractile filaments for cytokinesis low duty ratio non processive, myosin VI minus end directed for endocytosis. Therefore myosin V primary dedicated motor for processive intracellular cargo transport along actin cables and cortical networks.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 17: Myosin V and Cargo Transport.

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Cytoskeleton

Actin filaments interact with which motor protein for intracellular transport?

Two distinct superfamilies of cytoskeletal motors evolved to walk along filaments using ATP hydrolysis. Myosin motors share N terminal head motor domain containing actin binding interface including helix turn helix, cardiomyopathy loop, P loop ATPase, Switch I II for gamma phosphate sensing and lever arm with IQ motifs binding calmodulin light chains. Upon interaction with F actin 7 nanometer helical filament, head undergoes conformational cycle releasing phosphate driving swing of lever arm producing force along filament. Diverse isoforms specialize: myosin II non processive forming bipolar filaments for contraction, myosin V processive dimer for cargo, myosin I single headed tension sensor. In contrast dynein heavy chain AAA plus ring and coiled coil stalk binds microtubule 25 nanometer tubule, kinesin motor domain with tubulin binding loops moves along protofilaments. Nexin is non motor linker between doublet microtubules in axoneme. Therefore actin filament based intracellular transport and contractility specifically require myosin family as dedicated actin associated motor providing directional movement.

Ref: Alberts et al., Molecular Biology of the Cell, 7th ed., Chapter 16: Myosin Motors and Actin Filaments.

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Protein Sorting and Trafficking

The vesicle coat assembly process is initiated by:

Coat assembly regulated spatiotemporally to occur only at correct donor membranes and when cargo present ensuring energy not wasted. Initiating event activation of small GTPases Sar1 and ARF families from GDP cytosolic to GTP membrane state. Sar1 activated by Sec12 GEF integral ER protein exchanges GDP for GTP exposing amphipathic helix embedding ER leaflet inducing curvature recruiting Sec23-Sec24 heterodimer. Similarly ARF1 activated by GBF1 at ERGIC cis-Golgi and BIG1/2 at TGN upon membrane recruitment exposes myristoylated helix plus effector binding switch regions recruiting COPI coatomer heptamer or AP1/GGA clathrin adaptors beginning lattice nucleation. GTPases serve as membrane-anchored nucleation points; GDP form cytosolic inactive unable to bind coats. Direct clathrin triskelia binding membrane without adaptor or GTPase does not occur physiologically. ATP hydrolysis by NSF drives cis-SNARE disassembly post-fusion not coat initiation. Cargo phosphorylation may modulate adaptor affinity via casein kinase but nucleation itself requires active GTPase switch explaining sensitivity to fungal metabolite brefeldin A stabilizing abortive ARF-GDP-GEF complex and to non-hydrolyzable GTPgammaS locking coats on membranes preventing recycling and fusion competence.

Ref: Lee & Miller, Annu Rev Cell Dev Biol: Sar1 and ARF GTPases initiate coat assembly.

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Protein Sorting and Trafficking

What is the function of SNAP-25 in vesicle fusion?

Assembly of heteromeric SNARE core provides mechanical work for fusion in neurons, endocrine cells and constitutive secretion. Proteins classified into Q-SNAREs supplying glutamine at central zero ionic layer and R-SNAREs supplying arginine. Target plasma membrane harbors two Q proteins: syntaxin1 integral with single Q helix preceded by Habc regulatory domain clamped by Munc18 and SNAP-25 peripheral anchored via palmitoylated linker contributing two Q helices lacking transmembrane domain together contributing three Q motifs collectively t-SNARE. Vesicle membrane supplies R protein synaptobrevin VAMP2 with single R helix. Upon calcium entry triggering synaptotagmin complexin clamp released allowing N-to-C zippering forming parallel four-helix bundle extremely tight SDS resistant releasing about 65 kT energy deforming membranes into fusion pore. SNAP-25 does not act as kinase phosphorylating cargo nor initiate coat disassembly nor degrade misfolded proteins; role purely structural providing two helices. Botulinum toxins A, C, E cleave SNAP-25 or VAMP abolishing regulated exocytosis proving absolute requirement as t-SNARE component essential for neurotransmission.

Ref: Jahn & Scheller, Nat Rev Mol Cell Biol: SNAP-25 as Qbc t-SNARE in fusion.

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Protein Sorting and Trafficking

What is the function of the SNARE complex?

Vesicle fusion fidelity beyond tethering relies on SNARE proteins providing thermodynamic driver for bilayer merger overcoming hydration repulsion. Vesicle membranes contain R-SNAREs like synaptobrevin VAMP2 with single motif and C-terminal anchor, while target membranes harbor Q-SNAREs syntaxin family with Habc regulatory domain and SNAP-25 family contributing two motifs anchored by palmitoylation. When opposed membranes brought within few nanometers by Rab tethers and multisubunit complexes, oppositely oriented SNARE motifs assemble as parallel four-helix coiled coil from N-terminus to C-terminus zippering releasing substantial free energy. Formation pulls membranes into point contact inducing hemifusion stalk then fusion pore opening delivering content efficiently. Specificity arises from compatible QabcR combinations respecting ionic central layer; neuronal exocytosis uses syntaxin1-SNAP25-VAMP2, ER-Golgi uses Sed5-membrin-Bet1-Bos1-Sec22, endosome uses syntaxin7-8-Vti1b-VAMP8. They do not mediate budding or ATP hydrolysis or clathrin recruitment; those involve coats and GTPases. After fusion cis complex awaits disassembly by NSF ATPase AAA family for reuse throughout secretory pathway.

Ref: Jahn & Scheller, Nat Rev Mol Cell Biol 2006: SNARE four-helix bundle drives membrane fusion.

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Protein Sorting and Trafficking

What is the primary function of Rab proteins in vesicle transport?

Rab small GTPases orchestrate specificity along endomembrane system as largest branch of Ras superfamily cycling between cytosol and membrane. GDP-bound Rabs kept soluble by guanine nucleotide dissociation inhibitor GDI that shields geranylgeranyl tails. Upon recruitment by specific guanine nucleotide exchange factors TRAPP, DENND, Mon1-Ccz1 at target organelles, they exchange GDP for GTP exposing N-terminal amphipathic helices and prenyl anchors firmly embedding in bilayer. GTP conformation exposes switch regions binding diverse effectors: long coiled-coil golgins like GM130, p115, tethering complexes CORVET, HOPS, Dsl1, GARP, TRAPPII, and motors kinesin, dynein, myosin Vb for movement along microtubules and actin. Effectors mediate vesicle capture at up to 200 nm bringing vesicles close for SNARE pairing. After fusion, TBC domain GAPs stimulate GTP hydrolysis returning Rab to GDP and GDI extraction for another cycle. Humans encode over 60 Rabs marking distinct compartments: Rab1 ER-Golgi, Rab5 early endosomes, Rab7 late, Rab11 recycling. They do not directly fuse membranes nor hydrolyze ATP nor degrade cargo.

Ref: Alberts et al., MBC, Chapter 13: Rab GTPases as vesicle tethering and targeting regulators.

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