Myosin dynamics in live Dictyostelium cells

Area of Science:

• Cell Biology
• Biochemistry
• Molecular Biology

Background:

• Conventional myosin is crucial for cellular processes like cytokinesis and migration.
• Understanding myosin's role in nonmuscle cells requires functional assays.
• The Dictyostelium discoideum system offers a unique model for studying protein function in vivo and in vitro.

Purpose of the Study:

• To create and validate a functional green fluorescent protein (GFP)-tagged myosin heavy chain in Dictyostelium discoideum.
• To investigate the in vivo localization and in vitro biochemical activity of GFP-myosin.
• To explore the role of conventional myosin in cellular protrusions.

Main Methods:

• Constructing a fusion protein of GFP and Dictyostelium discoideum myosin heavy chain (GFP-myosin).
• Assessing the rescue of myosin null cell defects by expressing GFP-myosin.
• Purifying GFP-myosin and evaluating its ATPase activity and in vitro motility.
• Microscopic analysis of GFP-myosin localization in live cells during cytokinesis and migration.

Main Results:

• GFP-myosin expression fully rescues defects in myosin null cells.
• Purified GFP-myosin demonstrates biochemical and motility properties identical to wild-type myosin.
• GFP-myosin localizes to the cleavage furrow during cytokinesis and the posterior cortex during migration.
• A novel, transient accumulation of GFP-myosin is observed in the tips of retracting pseudopods.

Conclusions:

• GFP-myosin serves as a reliable reporter for conventional myosin function in Dictyostelium.
• Conventional myosin is essential for cytokinesis and cell migration dynamics.
• Myosin's role extends to the regulation of pseudopod dynamics, challenging previous assumptions about cellular protrusions.