Cell Biology JoVE (Open Access) Citable · DOI

Identification of Kinesin-1 Cargos Using Fluorescence Microscopy

Name: Identification of Kinesin-1 Cargos Using Fluorescence Microscopy
Uploaded: 2026-07-14
Duration: 8 min 6 s
Description: Fluorescence microscopy is employed to identify Kinesin-1 cargos. Recently, the heavy chain of Kinesin-1 (KIF5B) was shown to transport the nuclear ...

DOI: 10.3791/53632-v

What you'll learn

✓ Perform transfection of mammalian cells with motorless Kinesin-1 constructs
✓ Detect motor protein and cargo aggregation via fluorescence microscopy
✓ Apply cargo identification strategy to characterize motor protein interactions
✓ Interpret immunofluorescence imaging to identify kinesin-1 binding partners

Protocol

Biopharma Insights Here, a protocol is presented to identify Kinesin-1 cargos. A motorless mutant of the Kinesin-1 heavy chain (KIF5B) aggregates in the cytoplasm and induces aggregation of its cargos. Both aggregates are detected under fluorescence microscopy. A similar strategy can be employed to identify cargos of other motor proteins.

Difficulty

advanced

Total time

~3-4 days (cell culture preparation, transfection, fixation, immunostaining, imaging)

Model organism

Mammalian cultured cells (unspecified)

Biosafety

BSL-1

Steps

Prepare and culture mammalian cells for microscopy

Set up cell culture conditions appropriate for immunofluorescence microscopy. Plate cells at suitable density on coverslips or imaging dishes to achieve 50-70% confluency for transfection.

▶ 00:39

Transfect cells with motorless Kinesin-1 constructs

Prepare transfection complex containing motorless KIF5B mutant plasmid and deliver into cultured mammalian cells using standard transfection reagents. Allow cells to express construct for 24 hours.

▶ 01:32

Perform live imaging and immunofluorescence staining

Acquire live cell fluorescence microscopy images, then fix cells and perform indirect immunofluorescence using antibodies against Kinesin-1 and candidate cargo proteins to detect co-localized aggregates.

▶ 03:08

Analyze aggregation patterns via fluorescence microscopy

Image fixed and stained cells to visualize motor protein and cargo aggregate co-localization in the cytoplasm. Confirm cargo identification through spatial overlap of fluorescent signals at motorless Kinesin-1 aggregation sites.

▶ 05:40

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