Oliver I. Wagner, PhDAssistant Professor

National Tsing Hua University

Institute of Molecular & Cellular Biology

College of Life ScienceLaboratory course: Model organism C. elegansWeek 4:

What is trafficking?How is cargo transported?Motor-cargo specificitiesStudying trafficking using kymograph analysis

Trafficking in C. elegans neuronsVesicles and mitochondria move along actin or microtubule tracks attached to molecular motors as myosins, kinesins and dynein

v20-02-vesicle_transport.mov

Axonal transport of vesicles+-+-+- Synaptic vesicles and mitochondria are transported via kinesins from the cell body of the neuron to the termini (growth cone) The molecular motor dynein transports them backMitochondria(growth cone)synaptic vesicle

3D EM image of a mitochondrion(computer-generated from series of 2D EM images)Mitochondria arethe energy factoriesof the cell

Model of kinesin-based vesicle transport Kinesins bind via their motor domain to microtubules while the tail (cargo) domain is connected to the vesicle The vesicle connection is mediated by kinesin receptor proteins (linker proteins)

DendritesKinesin receptor control cargo attachmentsHirokawa and Takemura, 2005, Nat Rev Neurosci.Axon

Kinesin superfamily proteins (KIF) KIF1A is a monomeric kinesin: in C. elegans it is called UNC-104 It is the main synaptic vesicle transporter in neuronsThe mechanisms of kinesin I movement on microtubules is well known

16_7.mov

KIF1A knockout mice: defect in synaptic precursor transport and neuronal cell deathReduction in the density of synaptic vesicles in nerve terminals, accumulation of vesicles in the cell bodyKIF1A plays a critical role in the development of neuropathies resulting from impaired axonal transportWT wt/kif1a kif1a/kif1a(Yonekawa, JCB, 1998)

Dynein alone cannot attach to vesicles or mitochondria: it needsanother helper named dynactinDYNEINDYNACTIN

Dynactin is an adaptor to connect dynein to the vesicle and the microtubuleVesicleJoseph Roland 2002+-

Vale, 2003, CellThe motor toolbox for intra-cellular transport Motor domains = blue Cargo binding domains = purple

Taken from: Cell Biology, Pollard & EarnshawSynaptic vesicles move bidirectional: coordinated activity of antagonistic motors?

or tug-of-war between antagonistic motors?

Determination of motor activity by analyzing motility of UNC-104::GFP particles- movie length about 5 min.- width of neuron about 150 nm bidirectional velocity of 1 m/s fast axonal cargo transport

Living worm

The paper iscontinuously moving.

A stable spot in the axon remains as a line on the paper.

A moving spot will leave an individual trace on the paper.txA Kymograph is the translation of a moving spot, on a line in one direction, into a two dimensional projection area with time and distance.movieKymograph=> with time and distance we can calculate velocity, pausing, run length etc.

Translation of a particle movement from a movie-sequence into a kymograph

Example of data evaluation using the kymograph technique

In living wormsIn primary C. elegans neurons

Isolated primary C. elegans neuronsCurrent research example

*JIPs = scaffolding proteins of the c-Jun amino (N)-terminal kinase (JNK) signalling pathwayKAP3 = kinesin superfamily-associated protein 3APP = amyloid precursor proteinAPOER2 = apolipoprotein E receptor 2KLC = kinesin light chainAMPA = -amino-3-hydroxy-5-methyl-4-isoxazole propionic acidGRIP1 = glutamate receptor-interacting protein 1NMDA = N-methyl-D-aspartate

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