FVIII Trafficking Dynamics Across Subcellular Organelles Using CRISPR/Cas9 Specific Gene Knockouts

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Factor VIII (FVIII) secretion is crucial for hemostasis and is influenced by endoplasmic reticulum (ER) chaperones like Calnexin (CANX) and Calreticulin (CALR).
• Gamma-aminobutyric Acid Receptor-associated proteins (GABARAPs) have been previously implicated in modulating FVIII secretion.
• Understanding FVIII intracellular trafficking is key for developing effective FVIII replacement, cell, and gene therapies.

Purpose of the Study:

• To investigate the intracellular dynamics of FVIII secretion using CRISPR/Cas9 knockout models.
• To elucidate the roles of ER chaperones (CANX, CALR) and GABARAP proteins in FVIII trafficking.
• To assess the impact of cellular pathway modulators on FVIII secretion and localization.

Main Methods:

• Generation of single and double CRISPR/Cas9 knockout (KO) HEK293 cell lines expressing FVIII for CANX, CALR, and GABARAP proteins.
• Manipulation of cellular pathways using Brefeldin A (BFA), Chloroquine (CQ), a Rab7 inhibitor, and glucose starvation.
• Assessment of FVIII secretion via a two-stage chromogenic assay and intracellular distribution using immunofluorescence (IF) microscopy.

Main Results:

• Distinct effects of individual protein knockouts on FVIII trafficking were observed.
• FVIII phenotypes clustered in GABARAP^KO, CANX^KO, and CALR^KO cells, including double KO combinations.
• FVIII colocalization with endomembrane components (Rab8, Rab7, VAMP8) at the trans-Golgi indicated involvement of additional trafficking factors.

Conclusions:

• CANX, CALR, and GABARAPs play significant roles in regulating FVIII intracellular trafficking.
• The study highlights the complex interplay of chaperones and trafficking proteins in FVIII secretion.
• Findings provide new insights into FVIII molecular mechanisms, potentially informing future therapeutic advancements.